Novel scavenger receptors

ABSTRACT

Novel scavenger receptors having an SR structure and a collectin-like structure are provided, which can be utilized in the elucidation of mechanisms of macrophage and basic immunity; in the elucidation of mechanisms of the development of a wide variety of diseases such as arteriosclerosis, diabetic complications and Alzheimer&#39;s disease, hyper β-lipoproteinemia, hypercholesterolemia, hypertriglyceridemia, hypo α-lipoproteinemia, transplantation, atherectomy, post angiogenic restenosis, bacterial infections; in the diagnostic, prophylactic and therapeutic methods thereof; and in the development of reagents and drugs for the same. The novel scavenger receptors include proteins comprising an amino acid sequence set out in SEQ ID NO: 2, 4 or 24 or proteins having equivalent properties to the same, or derivatives or fragments thereof as well as isolated polynucleotides comprising a nucleotide sequence encoding these proteins, and related molecules such as antibodies, antagonists and the like. Also disclosed are methods for the treatment using the same.

FIELD OF THE INVENTION

[0001] The present invention relates to isolated human and mouse novelscavenger receptors (herein referred to as “hSRCL-P1” and “mSRCL-P1”respectively, or merely as “SRCL-P1” when discrimination is notintended), genes and proteins, the homologues, mutants, modified formsand polymorphic variants thereof (these are collectively referred to as“derivatives”), fragments thereof (hereinafter collectively referred toas “SRCL-P1s” for all of these), and the detection thereof. The presentinvention further relates to compositions which comprise SRCL-P1s forpharmaceutical use, diagnostic use and research use, and methods for theproduction and use of the same. Additionally, the present inventionrelates to agonists and antagonists of SRCL-P1s proteins, as well asmethods for screening drugs using SRCL-P1s. Moreover, the presentinvention relates to expression vectors comprising SRCL-P1s gene,transformed cells that were transformed with the expression vector,antibodies to SRCL-P1 protein, and cells that produce the antibody.

BACK GROUND OF THE INVENTION

[0002] Pathological features in lesions at an early stage ofatherosclerosis involve the event of increase of foam cells in arterywalls. Scavenger receptors (hereinafter abbreviated as “SR”) that arepresent on a cell membrane of a macrophage (Krieger, M. et al., Annu.Rev. Biochem., 63, 601-637, 1994) lack negative feed back regulation bycholesterol, alien from LDL receptors. Thus, the receptor itself changesinto foam cells through actively incorporatingsh modified LDL (lowdensity lipoprotein that is a complex of cholesterol and a lipoprotein)to accumulate beneath the vascular endothelial cells. Therefore,macrophages and SRs thereof have been believed to play important rolesin the establishment of pathosis of atherosclerosis (Brown, M. S. etal., Nature, 343, 508-509, 1990; Kurihara, Y. A. et al., Current Opinionin Lipidology, 2, 295-300, 1991; Krieger, M., TIBS, 17, 141-146, 1992;Krieger, M. et al., J. Biol. Chem., 268(7), 4569-4572, 1993).

[0003] Continuous hyperglycemia in a living body resulting from diabetescauses nonenzymatic glycation of various proteins, thereby leading theproduction of Maillard reaction-advanced end products (AGE:advancedglycation end products), which are final products in a glycation processvia a Schiff base and an Amadori compound. AGE having an injuriousaction on cells adversely affects through the binding to macrophages,vascular endothelial cells, hepatic cells, renal mesangium cells and thelike via AGE receptors. For example, it is known that secretion ofcytokines such as TNF (Tumor Necrosis Factor), IL-1 (Interleukine-1) andplatelet derived growth factor (PDGF) is accelerated upon binding of AGEto a macrophage, thereby causing cell injuries characteristic todiabetic complications. SR is believed to participate profoundly indiabetic complications such as diabetic nephropathy, diabeticretinopathy, diabetic neuropathy, on the basis of the findings that SRis one of the receptors involving in incorporation and degradation ofAGE (Araki, N. et al., Eur. J. Biochem., 230, 408-415, 1995; Suzuki, H.et al., Circulation, 92, 1-428, 1995), and that degradative activity ofAGE is lowered to a level of third in an SR-double knockout mouse.Further, when an excessive AGE albumin is administered to a rat, AGE wasfound to deposit in kidney, thereby developing and glomerulosclerosis(Vlassara, H. et al., Proc. Natl. Acad. Sci. USA, 91, 11704-11708,1994). Accordingly, SR, which recognizes AGE, is anticipated toprofoundly involve in glomerulosclerosis.

[0004] In addition, SR is believed to involve in Alzheimer's disease.Pathological features of Alzheimer's disease concern senile plaques thatare deposits of β-amyloid. β-amyloid has been reported to activatemicroglia cells via SRs that are expressed on the microglia cells togenerate active oxygen, leading to the expression of neurotoxicity(Nature, 382, 716-719, 1996).

[0005] Examples of ligand for SRs include: ligands having negativecharge, e.g., modified LDL such as acetylated LDL (AcLDL), oxidized LDL(OxLDL) and the like, modified proteins such as maleylated BSA and thelike, quadruple helical nucleic acids such as polyinosinic acids and thelike, polysaccharides such as dextran sulfate and fucoidane and thelike, acidic phospholipids such as phosphatidylserine,phosphatidylinositol and the like, endotoxin (LPS), AGE, senile cellsapoptotic cells, and the like, although differences in specificitythereof may exist depending on the differences of molecular species ofSRs. Additionally, SR is believed to play an important role in removalof foreign substances, metabolic decomposition products and the like,because SR extensively recognizes various modified substances and a widevariety of foreign substances such as viruses in a living body (Hampton,R. Y. et al., Nature, 352, 342-344, 1991; Tokuda, H. et al., Biochem.Biophys. Res. Commun., 196(1), 8-24, 1993; Pearson, A. M. et al., J.Biol. Chem., 268, 3546-3554, 1993; Dunne, D. W. et al., Proc. Natl.Acad. Sci. USA, 91, 1863-1867, 1994; Freeman, M. W. Current Opinion inLipidology, 5, 143-148, 1994).

[0006] SRs have been expressed in hepatic sinusoidal endothelial cells(Eskild, W. et al., Elsevier Biomedical N.Y., 255-262, 1982), vascularendothelial cells (Baker, D. P. et al., Arteriosclerosis, 4, 248-255,1984; Bickel, P. E. et al., J. Clin. Invest., 90, 1450-1457, 1992),blood smooth muscle cells (Pitas, R. E. et al., J. Biol. Chem., 265,12722-12727, 1990; Bickel, P. E. et al., J. Clin. Invest., 90,1450-1457, 1992), fibroblasts (Pitas, R. E. et al., J. Biol. Chem., 265,12722-12727, 1990), and the like as well as in macrophages. Further, SRshave been classified into SRA, SRB, SRC (Peason, A. et al., Proc. Natl.Acad. Sci. USA, 92, 4056-4060, 1995), FcγRIIB2 (Stanton, L. W. et al.,J. Biol. Chem., 270, 22446-22451, 1992) and macrosialin (CD68)(Ramprasad, M. P. et al., Proc. Natl. Acad. Sci. USA, 92, 9580-9584,1995), human vascular endothelial OxLDL receptor (LOX-1: lectin-likeoxidized LDL receptor) (Sawamura, T. et al., Nature, 386, 73, 1997).Moreover, SRA has been classified into SR-AI and SR-AII (Kodama, T. etal., Nature, 343, 531-535, 1990), and MARCO (a novel macrophage receptorwith collagenous structure) (Elomaa, O. et al., Cell, 80, 603-609,1995); SRB has been classified into CD36 (Endemann, G. et al., J. Biol.Chem., 268, 11811-11816, 1993) and SR-BI (Acton, S. L. et al., J. Biol.Chem., 269, 21003-21009, 1994).

[0007] SR-AI and SR-AII are homotrimers, which are of inside-out typetransmembrane proteins of which N-terminus resides within the cell. Theprotein is structurally revealed to have several domains such as acollagen-like domain, α-helical coiled coil domain and a cysteine-richdomain, and the like in its extracellular portion (Rohrer, L. et al.,Nature, 343, 570, 1990; Matsumoto, A. et al., Proc. Natl. Acad. Sci.USA, 87, 9133, 1990). The collagen-like domain has a structurecharacteristic in collagen, (Gly-Xaa-Yaa)n, wherein Xaa and Yaa may beany one of amino acid residues, and this domain functions as aligand-binding site. The α-helical coiled coil domain is a dexiotropichepted repeat which turns two times at every seven amino acids, namelyhaving a structure of α-helical coiled coil. The three polypeptides forma homotrimer with hydrophobic amino acids such as leucine and isoleucinethat are present at every seven amino acids being directed to inside ofthe molecule, whilst having polar amino acids and carbohydratechain-binding site outside thereof (leucine zipper). Roles of thedomains involve retention of the homotrimer structure, as well asbinding to the ligands such as modified LDL to incorporate them into thecells, and changing a tertiary structure of the receptor depending ondecrease of pH in endosome, finally resulting in the dissociation of theligands.

[0008] Intracellular domain of the protein has a tight turn structure,which is characteristically found in an endocytotic signal, similarly tothe structures including NPXY sequence found in LDL receptors or insulinreceptors and YXRF sequence found in transferrin receptors. It issuggested that endocytosis may be suppressed when these sequences aredeleted.

[0009] SR-A1 and SR-A2 arise from alternative splicing of mRNA coding acysteine-rich domain. SR-AI has 110 amino acids corresponding to thedomain, whilst SR-AII has corresponding 17 amino acids. SR-AI and SR-AIIare expressed in at least peripheral macrophage derived from monocyte,pulmonary alveolus macrophage and hepatic Kupffer cell. It is revealedthat they participate in a host defense system in a living body, forexample, arteriosclerosis, Calcium ion-independent cell adhesion and thelike (Krieger, M. et al., Annu. Rev. Biochem., 63, 601-637, 1994; Wada,Y et al., Ann. N.Y. Acad. Sci., 748, 226-239, 1995; Fraser, I. P. etal., Nature, 364, 343, 1993). Further, OxLDL is present withinmacrophages of arteriosclerosis foci. In addition, SR-AI and SR-AII areabundantly expressed on the cell membrane of macrophage, and theelevation of blood lipoprotein by lipid absorption is suppressed in atransgenic mouse for SR-AI. Accordingly, it is envisaged that SR-AI andSR-AII play important roles in incorporation of OxLDL.

[0010] To the contrary, although MACRO classified into SRA has a similarstructure as that of SR-AI, it has no α-helical coiled coil domain,which is characterized by having a long collagen-like domain. MACRO isexpressed in spleen macrophage, lymph node macrophage and the like,which is believed to function in a host defense mechanism againstbacterial infection in a living body taking into account of thespecificity of the ligands thereof.

[0011] Suzuki et al., successfully produced an SRA-knockout mousethrough the substitution of the fourth exon that is a common partbetween SR-AI and SR-AII with a neomycin resistant gene (Suzuki, H. etal., Nature, 386, 292-296, 1997). Immune disorder has been observed inthe SRA-knock out mouse in comparison with the wild type, and exhibits ahigh rate of infection with Listeria and herpes simplex virus. Inaddition, it is indicated that SRA participates in phagocytosis of Tcells having apoptosis occurred, and that the phagocytic capacity isreduced in the SRA-knockout mouse in comparison with the wild type(Platt, N. et al., Proc. Natl. Acad. Sci. USA, 93, 12456, 1996).Furthermore, in a double knockout mouse obtained by the mating of theSRA-knock out mouse and an apoE deficient mouse (Plump, A. S. et al.,Cell, 71, 343, 1992; Zhag, S. H. et al., J. Clin. Invest., 94, 937,1994) that is an animal model for arteriosclerosis, it is indicated thatthe area of arteriosclerosis foci is significantly smaller than that ofthe apoE deficient mouse (Suzuki, H. et al., Nature, 386, 292-296,1997).

[0012] Thus, SR can be utilized in the elucidation of functions ofmacrophage, the elucidation of mechanisms of development of varioustypes of diseases including for example, arteriosclerosis, diabeticcomplications and AD, hyper β-lipoproteinemia, hypercholesterolemia,hypertriglyceridemia, hypo α-lipoproteinemia, transplantation,atherectomy, post angiogenic restenosis and the like, as well asdiagnostic, prophylactic, therapeutic methods thereof, and in thedevelopment of reagents and drugs for the same. Accordingly, to findnovel molecular species belonging to this family can be the means tosolve the above-described problem to be solved.

[0013] Besides, a complement system that plays an important role in ahost defense mechanism is known to include: a classical pathway in whichan immunoglobulin serves as a recognition molecule followed by theactivation of C1 that is the first component of the complement; and analternative pathway in which C3, which is the third component of thecomplement, is directly coupled to foreign substances such as bacteria.In addition to these pathways of the complement activation, a lectinpathway was illustrated in which a mannose binding protein (hereinafterreferred to as “MBP”), which is a serum lectin, activates the complementsystem through the direct recognition of and coupling with acarbohydrate chain on the surface of the foreign substance, in recentyears (Sato, T. et al., Int. Immunol., 6, 665-669, 1994).

[0014] MBP is a C type lectin which specifically binds to mannose,N-acetylglucosamine and the like in the presence of Calcium ion, ofwhich structure comprises a collagen-like domain containing at least(Gly-Xaa-Yaa)n, and carbohydrate recognition domain (CRD). Similarly toMBP, lectins having a collagen-like domain and CRD are genericallycalled as collectin (Malhotora, R. et al., Eur. J. Immunol., 22,1437-1445, 1992), which include collectin-43 (CL-43), surfactant proteinA (SP-A), surfactant protein D (SP-D), bovine conglutinin (BKg) and thelike, in addition to MBP. Collectin has an opsonic activity, which isbelieved to participate in basal immunity against a variety ofmicroorganisms such as bacteria and viruses (Kawasaki, N. et al., J.Biochem., 106, 483-489, 1989; Ikeda, K. et al., J. Biol. Chem., 262,7451-7454, 1987; Ohta, M. et al., J. Biol. Chem., 265, 1980-1984, 1990;Summerfield, J. A. et al., Lancet, 345, 886, 1995).

[0015] These collectins are known to constitute from a basic structurecontaining characteristic domains such as (1) CRD and (2) collagen-likedomain and the like as shown in FIG. 1(a) (Malhortra et al., Eur. J.Immunol., 22, 1437-1445, 1992). This basic structure forms a subunitthrough composing a triple helix at the collagen-like domain, and thusthese subunits further form an oligomer structure such as trimer,tetramer, hexamer and the like.

[0016] Recently, collectins were suggested to participate innon-specific immune response, e.g., it was reported that for example,they are playing important roles in neutralizing and excluding variousmicroorganisms in infants having maternal antibodies from the mother ornonspecific defense systems which were insufficiently developed (Superet al., Lancet, 2, 1236-1239, 1989). Moreover, results of investigationare reported involving in roles of these collectins in the body defensesystem of a host, which for example, suggest that the host becomes moresusceptible to infections through the lowered concentration of MBP inblood resulting from genetic mutation of MBP (Sumiya et al., Lancet,337, 1569-1570, 1991). In addition, it was reported that serum MBPcontent shows a lowered level upon the failure of opsonization (Madsen,H. O. et al., Immuno genetics, 40, 37-44, 1994), whilst bacterialinfections readily occur (Garred, P. et al., Lancet, 346, 941-943,1995). Therefore, MBP may be believed to play important roles in animmune system.

[0017] The present inventors previously found that BKg and MBP inhibitinfections by H1 and H2 types influenzae A viruses as well as ahaemagglutination activity (Wakamiya et al., Glycoconjugate J., 8, 235,1991; Wakamiya et al., Biochem. Biophys. Res. Comm., 187, 1270-1278,1992). Thereafter, a cDNA clone encoding BKg was obtained, and therelevance between BKg and SP-D and the like has been also found (Suzukiet al., Biochem. Biophys. Res. Comm., 191, 335-342, 1993).

[0018] Likewise, collectins are substances to which usefulness in theelucidation of host defense mechanism and utilities as a biologicallyactive substance are expected. Thus, the finding of novel molecularspecies belonging to this family may greatly contribute in variousmedical fields and biological fields in addition to the therapy ofinfectious diseases.

DISCLOSURE OF THE INVENTION

[0019] The object of the present invention is to provide a novelscavenger receptor that can be utilized in the elucidation of mechanismsof macrophage and basic immunity; in the elucidation of mechanisms ofthe development of a wide variety of diseases such as arteriosclerosis,diabetic complications and Alzheimer's disease, hyper β-lipoproteinemia,hypercholesterolemia, hypertriglyceridemia, hypo α-lipoproteinemia,transplantation, atherectomy, post angiogenic restenosis, bacterialinfections; in the diagnostic, prophylactic and therapeutic methodsthereof; and for the development of reagents and drugs for the same.

[0020] Accordingly, the aspects provided by the present invention are asdescribed below.

[0021] (1) A protein comprising an amino acid sequence consisting of 742amino acids set out in amino acid position 1 to 742 of SEQ ID NO: 2, ora protein comprising an amino acid sequence set out in SEQ ID NO: 2having deletion, substitution or addition of one or several amino acidstherein and having an equal property to that of the protein comprisingan amino acid sequence set out in amino acid position 1 to 742 of SEQ IDNO: 2, or a derivative or a fragment thereof;

[0022] (2) An isolated polynucleotide comprising a nucleotide sequenceset out in nucleotide position 74 to 2299 of SEQ ID NO: 1, a nucleotidesequence encoding an amino acid sequence set out in amino acid position1 to 742 of SEQ ID NO: 2 or a fragment thereof, or a nucleotide sequencethat hybridizes to any one of said nucleotide sequences or nucleotidesequences complementary thereto under a stringent condition and encodesa protein having an equal property to that of the protein comprising anamino acid sequence set out in amino acid position 1 to 742 of SEQ IDNO: 2;

[0023] (3) A protein comprising an amino acid sequence set out in aminoacid position 1 to 618 of SEQ ID NO: 24, or a protein comprising anamino acid sequence set out in SEQ ID NO: 24 having deletion,substitution or addition of one or several amino acids therein andhaving an equal property to that of the protein comprising an amino acidsequence set out in amino acid position 1 to 618 of SEQ ID NO: 24, or aderivative or a fragment thereof;

[0024] (4) An isolated polynucleotide comprising a nucleotide sequenceset out in nucleotide position 74 to 1933 of SEQ ID NO: 23, a nucleotidesequence encoding an amino acid sequence set out in amino acid position1 to 618 of SEQ ID NO: 24 or a fragment thereof, or a nucleotidesequence that hybridizes to any one of said nucleotide sequences ornucleotide sequences complementary thereto under a stringent conditionand encodes a protein having an equal property to that of the proteincomprising an amino acid sequence set out in amino acid position 1 to618 of SEQ ID NO: 24;

[0025] (5) A protein comprising an amino acid sequence consisting of 742amino acids set out in amino acid position 1 to 742 of SEQ ID NO: 4, ora protein comprising an amino acid sequence set out in SEQ ID NO: 2having deletion, substitution or addition of one or several amino acidstherein and having an equal property to that of the protein comprisingan amino acid sequence set out in amino acid position 1 to 742 of SEQ IDNO: 2, or a derivative or a fragment thereof;

[0026] (6) An isolated polynucleotide comprising a nucleotide sequenceset out in nucleotide position 74 to 2299 of SEQ ID NO: 3, a nucleotidesequence encoding an amino acid sequence set out in amino acid position1 to 742 of SEQ ID NO: 2 or a fragment thereof, or a nucleotide sequencethat hybridizes to any one of said nucleotide sequences or nucleotidesequences complementary thereto under a stringent condition and encodesa protein having an equal property to that of the protein comprising anamino acid sequence set out in amino acid position 1 to 742 of SEQ IDNO: 2;

[0027] (7) A vector comprising a polynucleotide according to (2), (4) or(6);

[0028] (8) A transformed cell carrying a polynucleotide according to(2), (4) or (6) in a manner to allow the expression;

[0029] (9) A method for the production of a protein which comprises thestep of culturing a cell transformed with the polynucleotide accordingto (2) or (4), and collecting thus produced hSRCL-P1 protein;

[0030] (10) A method for the production of a protein which comprises thesteps of culturing a cell transformed with the polynucleotide accordingto (6), and collecting thus produced mSRCL-P1 protein;

[0031] (11) The method according to (9) or (10) wherein said cell isEscherichia coli, an animal cell or an insect cell;

[0032] (12) A transgenic non-human animal having an altered expressionlevel of SRCL-P1 gene;

[0033] (13) The transgenic non-human animal according to (12) whereinsaid SRCL-P1 gene is cDNA, genomic DNA or synthesized DNA encodingSRCL-P1;

[0034] (14) The transgenic non-human animal according to (13) whereinthe expression level is altered by causing the mutation at a geneexpression regulatory site;

[0035] (15) A knockout mouse wherein a function of mSRCL-P1 gene isdeficient;

[0036] (16) An antibody to the protein according to (1), (3) or (5), ora fragment thereof;

[0037] (17) The antibody according to (16), which is a polyclonalantibody, a monoclonal antibody or a peptide antibody.

[0038] (1 8) A method for the production of a monoclonal antibody to theprotein or the fragment thereof according to (1), (3) or (5) whichcomprises administering the protein or a fragment thereof according to(1), (3) or (5) to a warm-blooded animal other than human, selecting theanimal that exhibits an antibody titer, collecting a spleen or a lymphnode from the animal, fusing antibody-producing cells contained thereinwith myeloma cells to prepare a hybridoma that produces a monoclonalantibody;

[0039] (19) A method for quantitatively determining an SRCL-P1 proteinor a fragment thereof on the basis of an immunological binding betweenthe antibody according to (16) or (17) and the SRCL-P1 protein or afragment thereof;

[0040] (20) A method for detecting an SRCL-P1 protein or a fragmentthereof on the basis of an immunological binding between the antibodyaccording to (16) or (17) and the SRCL-P1 protein or a fragment thereof;

[0041] (21) An agonist that stimulates an activity of the proteinaccording to (1), (3) or (5);

[0042] (22) An antagonist that inhibits an activity or the activation ofthe protein according to (1), (3) or (5);

[0043] (23) A method for screening a drug wherein the protein accordingto (1), (3) or (5) is used;

[0044] (24) A drug which is obtained by the method for the screeningaccording to (23);

[0045] (25) A method for screening a drug for the treatment of apathological state involved in the accumulation of oxidized LDL, whichcomprises the step of identifying a candidate drug for the treatment ofa pathological state involved in the accumulation of oxidized LDL by aninhibitory ability of the candidate drug toward the binding between theprotein according to (1), (3) or (5) and oxidized LDL, which isevaluated by comparing the amount of binding between the protein andoxidized LDL in the presence and absence of the candidate drug;

[0046] (26) A drug obtained by the method for the screening according to(25);

[0047] (27) A method for the treatment of a pathological state involvedin the accumulation of oxidized LDL, which comprises the step ofinhibiting the binding between an SRCL-P1 protein or a fragment thereofand oxidized LDL using the drug according to (26);

[0048] (28) A pharmaceutical composition for the treatment of apathological state involved in the accumulation of oxidized LDLcomprising the drug according to (26);

[0049] (29) A method for screening a drug for the treatment of apathological state involved in the binding of AGE to cells, whichcomprises the step of identifying a candidate drug for the treatment ofa pathological state involved in the binding of AGE to cells by aninhibitory ability of the candidate drug toward the binding between theprotein according to (1), (3) or (5) and AGE, which is evaluated bycomparing the amount of binding between the protein and AGE in thepresence and absence of the candidate drug;

[0050] (30) A drug obtained by the method for the screening according to(29);

[0051] (31) A method for the treatment of a pathological state involvedin the binding of AGE to cells, which comprises the step of inhibitingthe binding between an SRCL-P1 protein or a fragment thereof and AGEusing the drug according to (30); and

[0052] (32) A pharmaceutical composition for the treatment of apathological state involved in the binding of AGE to cells comprisingthe drug according to (30);

BRIEF DESCRIPTOIN OF THE DRAWINGS

[0053]FIG. 1 is a schematic drawing illustrating a basic structure ofprincipal collectins reported heretofore and an overview of the protein.

[0054]FIG. 2 is a drawing illustrating a preceding half of an alignmentof amino acid sequences of three kinds of collectins reportedheretofore.

[0055]FIG. 3 is a drawing illustrating a latter half of similaralignment to that of FIG. 2.

[0056]FIG. 4(b) depicts drawings showing a nucleotide sequence read by asequencer and each primer employed for the sequencing of the novelscavenger receptor of the present invention; and FIG. 4(a) depicts adrawing showing ORF of the novel collectin obtained.

[0057]FIG. 5 is a drawing illustrating a manner how A: yeast, B: gramnegative bacteria (Escherichia coli), and C: gram positive bacteria(Staphylococcus aureus) specifically bind to cells that are expressinghSRCL-P1.

[0058]FIG. 6 is a drawing illustrating a manner how A: oxidized LDL, B:mannose, and C: AGE specifically bind to cells that are expressinghSRCL-P1.

[0059]FIG. 7 is a drawing illustrating a manner how yeast isincorporated into cells that are expressing hSRCL-P1.

[0060]FIG. 8 is a drawing illustrating a manner how hSRCL-P1 isexpressed in A: healthy human heart vascular endothelial cells, and B:mouse heart vascular endothelial cells.

BEST EMBODIMENT FOR CARRYING OUT THE INVENTION

[0061] The present inventors successfully cloned human and mouse novelSR. A collectin domain containing CRD is present at C-terminuses of thenovel SR (SRCL-P1), which is believed to participate in basic immunity,and the entire structure of the novel SR was similar to that of SRA, inparticular, SR-AI. More specifically, it was constituted at least from atransmembrane domain comprising a leucine zipper that has leucine unitsrepeated four times, an α-helical coiled coil domain, a collagen-likedomain, a neck domain, a CRD domain, starting from the N-terminuses.Three molecules having such a characteristic structure are envisaged toform a homotrimer through the formation of an α-helix at the coiled coildomain and the formation of a triple helix at the collagen-like domain.Further, the collagen-like domain is speculated to be positively chargedunder a condition of physiological pH. In addition, SRCL-P1 protein hadnumerous carbohydrate chain binding sites

[0062] The hSRCL-P1 gene and mSRCL-P1 gene used herein includepolynucleotides comprising a nucleotide sequence set out in SEQ ID NO: 1or 3, derivatives thereof (homologues, mutants, modified forms andpolymorphic variants), and fragments thereof, unless otherwise stated.Further, the hSRCL-P1 protein and mSRCL-P1 protein used herein compriseamino acid sequence set out in SEQ ID NO: 2 or 4, derivatives thereofand fragments thereof, unless otherwise stated. These may be derivedfrom natural sources, or artificially synthesized. The present inventionincludes whole of the substances as described above.

[0063] Examples of hSRCL-P1 include proteins comprising amino acids setout in SEQ ID NO: 24 (a mutant of the protein set out in SEQ ID NO: 1with deletion of a part of the collagen-like domain and the neck domain,i.e., amino acid residues of position 483 to 606), and examples ofmutants of the polynucleotide set out in SEQ ID NO: 1 include apolynucleotide set out in SEQ ID NO: 23 which encodes the protein setout in SEQ ID NO: 24.

[0064] Moreover, the present invention also involves amino acidsequences substantially similar to the amino acid sequence set out inSEQ ID NO: 2 or 4, and nucleotide sequences encoding amino acidsequences substantially similar to the amino acid sequence set out inSEQ ID NO: 2 or 4. Furthermore, proteins comprising these amino acidsequences are also involved. The amino acid sequence substantiallysimilar to the amino acid sequence set out in SEQ ID NO: 2 or 4 refersto the amino acid sequence having alteration such as substitution,deletion, addition and/or insertion of one or several amino acidstherein as long as the protein has an equal property to those of theprotein comprising an amino acid sequence set out in SEQ ID NO: 2 or 4,that is to say; activity, function and tertiary structure due to thestructure which comprises a transmembrane domain containing a leucinezipper structure, and α-helical coiled coil domain and collagen-likedomain, which are characteristic in SR. These may be derived fromnatural sources, or artificially synthesized.

[0065] Furthermore, the present invention also involves a nucleotidesequence set out in SEQ ID NO: 1 or 3 or a nucleotide sequencecomprising a fragment thereof, or a nucleotide sequence that canhybridize to a nucleotide sequence complementary thereto (hereinafter,referred to as “specified sequence”) under a stringent condition. Thestringent condition according to the present invention may involve acondition for example; incubating in a solution containing 5×SSC, 5%Denhardt's solution (0.1% BSA, 0.1% Ficoll 400, 0.1% PVP), 0.5% SDS, and20 μg/ml modified sermon sperm DNA at 37° C. overnight followed by awash with 2×SSC containing 0.1% SDS at room temperature. SSPE may beemployed in place of SSC. Thus resultant nucleotide sequence isspeculated to exhibit at least 50% or more homology with the specifiedsequence. Many of the proteins encoded by the nucleotide sequence thathybridize to the specified sequence under a stringent hybridizationcondition are believed to have an equal property to SRCL-P1 protein.Therefore, such proteins are also involved in the present invention aslong as they have an equal property to SRCL-P1 protein.

[0066] In particular, the amino acid sequence of hSRCL-P1 set out in SEQID NO: 2 (amino acid position 1 to 742) represents a protein consistingof 742 amino acids, and thus the nucleotide sequence encoding the sameconsists of 2226 nucleotides. Characteristic amino acid sequences suchas those of a leucine zipper domain, an α-helical coiled coil domain, acollagen-like domain, a neck domain, a CRD domain and the like werepresent in the sequence. That is to say, a leucine zipper domaindesignated by amino acid position 36-57, an α-helical coiled coil domaindesignated by amino acid position 72-426 (according to COILS Program) oramino acid position 81-431 (according to MultiCoil Program), acollagen-like domain designated by amino acid position 443-589, a neckdomain designated by amino acid position 590-606, a CRD domaindesignated by 607-742 and the like were present. Other domains includefor example, an extracellular domain designated by amino acid position63-742 (according to TMHMM1.0 program) or amino acid position 58-742(according to TMpred program), an intracellular domain designated byamino acid position 1-39 (according to TMHMM1.0 program) or amino acidposition 1-37 (according to TMpred program), a transmembrane domaindesignated by amino acid position 40-62 (according to TMHMM1.0 program)or amino acid position 38-57 (according to TMpred program), acollectin-like domain designated by amino acid position 443-742.Moreover, a C type lectin motif designated by amino acid position708-730 was also included. The nucleotide sequence encoding this proteinis set out in SEQ ID NO: 1.

[0067] The amino acid sequence of mSRCL-P1 (amino acid sequence 1-742)set out in SEQ ID NO: 4 represents a protein consisting of 742 aminoacids, and thus the nucleotide sequence encoding the same consists of2226 bases. Similarly to hSRCL-P1 set out in SEQ ID NO: 2,characteristic amino acid sequences such as a leucine zipper domain, anα-helical coiled coil domain, a collagen-like domain, a neck domain, aCRD domain, a C type lectin motif and the like were present in thesequence. The nucleotide sequence encoding this protein is set out inSEQ ID NO: 3.

[0068] Homologues used herein refer to nucleotide sequences or aminoacid sequences that bear high homology, which are homologous at least50% or more, preferably 70% or more, more preferably 90% or more. Whendeletion or insertion is present in the sequence, homologous search maybe conducted which allows for gap junction. For example, the search maybe performed using a procedure of multiple alignment (trade name: SODHO,Fujitsu Limited). In addition, as the algorithm for searching homology,Smith-Waterman algorithm, which is the most accurate, may be employed.Alternatively, FASTA or BLAST may be also utilized via the Internet.

[0069] Mutants used herein include for example, those resulting fromallele, Single Nucleotide Polymorphism (SNP) and the like. Furthermore,the nucleotide sequence of the present invention may also include themutated nucleotide sequences derived from the changes in the range ofdegeneracy of the codon. Partial alteration of the codon of a nucleotidesequence may be achieved according to a routine method using such sitedirected mutagenesis methods as those in which a primer is employedconsisting of a synthesized oligonucleotide that encodes the desiredalteration (Mark, D. F. et al., Proc. Natl. Acad. Sci. USA., 81, 5662,1984). Thus resultant artificial genetic mutants are also involved inthe nucleotide sequence of the present invention. Furthermore, themutated amino acids translated by the mutated codons have preferablysimilar properties to those of the normal amino acid even in the casewhere the mutation is beyond the range of degeneracy of the codon. Themutation may preferably be as follows, which are among amino acidshaving similar properties, functions, characteristics and the like, forexample: the mutation among aliphatic amino acids such as alanine,valine, leucine and isoleucine; the mutation among neutral amino acidssuch as glycine, alanine, serine, threonine, valine, leucine,isoleucine, cysteine, methionine, phenylalanine, tyrosine, proline,tryptophan, asparagines and glutamine; the mutation among acidic aminoacids such as aspartic acid and glutamic acid; the mutation among basicamino acids such as arginine, lysine and histidine; the mutation amongserine and threonine, having a hydroxyl group; the mutation amongphenylalanine and tyrosine, having an aromatic ring; and the like. Theseartificially or naturally mutated proteins are also included in theprotein of the present invention. For the artificial mutation,site-directed mutagenesis may be caused using a PCR method, andalternatively, other known methods may be used to cause mutation at anyoptional site.

[0070] Modified forms used herein may be prepared using conventionaltechniques, for example, by acetylation, acylation, ADP-ribosylation,amidation, myristoylation, glycosylation, hydroxylation,phosphorylation, sulfation, formylation, methylation,polyethyleneglycolation, lipid coupling, nucleotide coupling, metalcoupling (calcium addition and the like), fusion with other protein(albumin and the like), dimerization, and the like. For example, sinceglycosylation does not occur in Escherichia coli as a host, theexpression may be conducted in eucaryotic cells when glycosylation isintended. Insect cells may be also used because glycosylation proceedspost-translationally, similarly to in mammalian cells.

[0071] Polymorphic variants used herein involve for example,polymorphisms caused by structural or conformational differences inchromosomal DNA, polymorphisms resulting from a change of a gene intoits allelic gene, or the like. In general, genes of eucaryotic cellsoften exhibit polymorphic event, and this event may lead to thesubstitution of one or more amino acid(s) whilst the activity of theprotein may be retained. Therefore, any of the genes encoding a proteinobtained by artificially modifying the gene encoding any of the aminoacid sequence set out in SEQ ID NO: 2 or 4 is involved in the presentinvention as far as the protein has a characteristic function of a geneof the present invention. In addition, any of the proteins comprisingartificially modified amino acid sequence set out in SEQ ID NO: 2 or 4is involved in the present invention as far as it has a property of aprotein of the present invention. The modification is construed asinvolving substitution, deletion, addition and/or insertion.

[0072] Fragments used herein refer to any optional fragments derivedfrom the amino acid sequence of SRCL-P1 described above, which includefor example, an extracellular domain, an intracellular domain, atransmembrane domain, a leucine zipper domain, an α-helical coiled coildomain, a collagen-like domain, a neck domain, a CRD domain, acollectin-like domain, a hydrophobic domain (a neck domain, atransmembrane domain and the like), a hydrophilic domain (domains otherthan hydrophobic domains), and the like, as well as fragments obtainedby the fusion of these fragments. For example, in the amino acidsequence of hSRCL-P1 set out in SEQ ID NO: 2, the fragments included maybe: a fragment comprising amino acids of from position 58-63 to position742 that form a soluble receptor but lacks a transmembrane domain; afragment comprising amino acids of from position about 1 to 606 thatform a transmembrane scavenger receptor but lacks a CRD domain; afragment comprising amino acids of from position about 36 to position426-431 that form a soluble scavenger receptor containing a leucinezipper and an α-helical coiled coil domain; and a fragment comprisingamino acids of from position 1 to 589 which lacks a CRD domain and aneck domain.

[0073] Method for Obtaining SRCL-P1 Gene

[0074] A SRCL-P1 gene according to the present invention may be thoseobtained through any methods. For example, the nucleotide sequenceencoding SRCL-P1 of the present invention can be obtained by preparingmRNA from the cells that are expressing the protein, and altering itinto a double stranded DNA by a conventional technique. For thepreparation of mRNA, guanidine isothiocyanate calcium chloride method(Chirwin, et al., Biochemistry, 18, 5294, 1979) and the like can beemployed. For the preparation of poly(A)⁺ RNA from total RNA, supportsbound with oligo(dT), for example, affinity chromatography in whichsepharose or latex particles are used, can be employed. Double strandedcDNA can be obtained by using thus obtained RNA described above as atemplate to treat with reverse transcriptase using oligo(dT) that iscomplementary to poly(A) chain present at 3′-terminus, or a randomprimer or a synthesized oligonucleotide corresponding to a part of theamino acid sequence of SRCL-P1 as a primer (Mol. Cell Biol., 2, 161,1982; Mol. Cell Biol., 3, 280, 1983; Gene, 25, 263, 1983); and treatingthus resulting cDNA with for example, E. coli RNaseH, E. coli DNApolymerase 1, E. coli DNA ligase to alter into the DNA chain. A cDNAlibrary can be produced by incorporating this cDNA into a plasmidvector, phage vector, cosmid vector to transform E. coli, or bytransfecting it into E. coli following in vitro packaging.

[0075] The plasmid vector that can be used herein is not particularlylimited as long as it can be replicated and maintained in the host.Phage vector is not also particularly limited as long as it canproliferate in the host. Cloning vectors include, for example, pBR322,pUC19, λgt10, λgt11 and the like. Moreover, upon subjecting toimmunological screening, the vector has preferably a promoter thatenables the expression of an SRCL-P1 gene in the host.

[0076] To incorporate cDNA into a plasmid, the method of Maniatis et al.(Molecular Cloning, A Laboratory Manual, second edition) and the likecan serve as a reference. Further, to incorporate cDNA into a phagevector, the method disclosed in Hyunh et al. (DNA cloning, a practicalapproach, 1, 49, 1985) and the like can serve as a reference.

[0077] As the method for introducing the expression vector describedabove into host cells, methods for example, transfection bylipopolyamine method, DEAE-dextran method, Hanahan method, lipofectinmethod, calcium phosphate method; microinjection, and electroporation(Molecular Cloning, A Laboratory Manual, second edition) and the likemay be involved. In vitro packaging can be readily effected by usingcommercially available kits (manufactured by Stratagene, or Amersham).

[0078] The method for the isolation of cDNA encoding SRCL-P1 proteinfrom a cDNA library prepared as described above may involve a generalmethod, which may be used in combination, for the screening of cDNA. Forexample, a probe labeled with ³²P is produced, and a clone containingthe desired cDNA can be screened by a colony hybridization method (Proc.Natl. Acad. Sci. USA, 72, 3961, 1975), or a plaque hybridization method(Molecular Cloning, A Laboratory Manual, second edition, Cold SpringHarbor Laboratory, 2, 108, 1989). Further, a clone may be selected by aPCR method. Additionally, the desired clone can be selected through useof an antibody that recognizes SRCL-P1 when a cDNA library is producedusing a vector that can express cDNA.

[0079] Furthermore, when an SRCL-P1 gene is isolated from cells thatexpress SRCL-P1 gene, for example, the expressing cells are dissolvedusing SDS or proteinase K, followed by a phenol treatment. Unwanted RNAis digested with ribonuclease. Thus resultant DNA is digested withrestriction enzyme, and the resulting DNA fragments are amplified usingphage or cosmid to produce a library. Thereafter, the desired clone isselected, and then an SRCL-P1 gene can be obtained.

[0080] The nucleotide sequence of the DNA obtained accordingly can bedetermined by a Maxam-Gilbert method (Proc. Natl. Acad. Sci. USA, 74,560, 1977) or a Sanger's method (Proc. Natl. Acad. Sci. USA, 74, 5463,1977). The SRCL-P1 gene can be obtained by excising from the clone asobtained above.

[0081] Through use of the primer synthesized on the basis of thenucleotide sequence of SRCL-P1, cloning can be also effected by a RT-PCRmethod using poly(A)⁺ RNA of the cells expressing SRCL-P1 as a template.Further, the desired cDNA can be also obtained by directly screening thecDNA library after producing/synthesizing a probe based on thenucleotide sequence of SECL-P1, not by way of the PCR. The gene of thepresent invention can be selected among the genes obtained by themethods described above through the verification of the nucleotidesequence of the gene. The gene of the present invention can be alsoproduced according to the conventional method in which chemicalsynthesis of a nucleic acid, e.g., phosphoimidite method (Mattencci, M.D. et al., J. Am. Chem. Soc., 130, 3185, 1981) or the like, is employed.

[0082] Method for Producing Expression Vector

[0083] The present invention also relates to a vector comprising anucleotide sequence of SRCL-P1s. The vector is not particularly limitedso far as it can express the SRCL-P1s protein, for example, a plasmidvector, an RNA vector, a DNA vector, a virus vector, a phage vector andthe like may be employed. Specifically, examples thereof includepBAD/His, pRSETA, pcDNA2.1, pTrcHis2A, pYES2, pBlueBac4.5, pcDNA3.1 orpSecTag2 manufactured by Invirtogen, pET or pBAC manufactured by NovagenCo., pGEM manufactured by Promega, pBluescriptII, pBS, Phagescript, pSGor pSV2CAT manufactured by Stratagene, or pGEX, pUC18/19, pBPV, pSVK3 orpSVL manufactured by Pharmacia Co.

[0084] The cDNA sequence of SRCL-P1s ligated to the expression vector isoperatively linked to a promoter. The promoter includes for example,phage λPL promoter, E. coli lac, trp, tac promoter, SV40 early and latepromoter, T7 and T3 promoter, retrovirus LTR promoter. Specifically, theprompter for use in eukaryotic cells include CMV promoter, HSV promoter,SV40 early and late promoter, retrovirus LTR promoter, RSV promoter,metallothionein promoter. In addition, the expression vector may containa marker to allow the selection of the transformed host, and anenhancer. Examples of the marker include dihydrofolate reductase gene,neomycin resistant gene, ampicillin resistant gene and the like.Examples of the enhancer include SV40 enhancer, cytomegalovirus earlyenhancer promoter, adenovirus enhancer and the like.

[0085] Method for Producing Transformed Cells

[0086] The present invention further provides transformed cells carryinga nucleotide sequence of the present invention to allow the expressionthereof by means of the vector as described above carrying thenucleotide sequence. The host cell for use as a transformed cell in thepresent invention may preferably include animal cells and insect cells,however, included may be any of the cells (microorganisms may be alsoincluded), which can express SRCL-P1s protein in the expression vectorof the present invention.

[0087] Exemplary animal cells and insect cells of the present inventionmay respectively include cells derived from human, or cells derived fromfly or silkworm (Bombyx mor). For example, CHO cells, COS cells, BHKcells, Vero cells, myeloma cells, HEK293 cells, HeLa cells, Jurkatcells, mouse L cells, mouse C127 cells, mouse FM3A cells, mousefibroblast, osteoblast, chondrocyte, S2, Sf9, Sf21, High Five™ cells maybe included. The microorganism according to the present inventioninclude Escherichia coli, Saccharomyces cerevisiae and the like. For theintroduction of a vector into such hosts, the method as described abovemay be employed.

[0088] In regard to SR pathway involved in the onset of arteriosclerosisand the like, SR-expressing cells of the present invention can be usedfor analyzing the specificity of modified LDLs that are incorporatedinto cells from this pathway. In addition, they are useful as models forthe analysis of incorporation of substances into the cells via areceptor. Moreover, the cells of the present invention can be used forscreening drugs in the process of developing therapeutic drugs ofarteriosclerosis, for example, depressants of LDL modification,inhibitors of acyl Co-A cholesterol acyltransferase (ACAT) activity, andthe like. Furthermore, they can be used for the manufacture of human SRprotein having a carbohydrate chain. They may be also employed inexperimental systems for the process of treating foreign substances ordenaturated substances via SR, or in systems for investigating infectionof B type viruses that cause infection concomitant with a modifiedalbumin.

[0089] Method for Obtaining Protein

[0090] The present invention also relates to a method for the productionof SRCL-P1 which comprises culturing a cell transformed with thenucleotide sequence of the present invention as set forth above, andharvesting thus produced SRCL-P1. Cell culture, isolation of theprotein, and purification may be carried out with conventionally knownmethods.

[0091] The protein of the present invention can be expressed as arecombinant fusion protein, which can be readily isolated, purified, andrecognized per se. The recombinant fusion protein is a protein expressedby adding an appropriate peptide chain to the N-terminal end and/orC-terminal end of a protein expressed from a nucleotide sequenceencoding the desired protein. In order to facilitate the purification ofthe expressed protein, the protein may be expressed as a fusion proteinhaving a signal for extracellular secretion. In addition, the proteincan be obtained from several kinds of sources such as cultured cells,cultured tissues, transformed cells and the like using conventionallyknown methods, for example, known purification methods including:salting out such as ammonium sulfate precipitation technique and thelike; gel filtration technique such as Sephadex and the like; ionexchange chromatographic technique; hydrophobic chromatographictechnique; dye gel chromatographic technique; electrophoresis technique;dialysis; ultrafiltration technique; affinity chromatographic technique;high performance liquid chromatographic technique; and the like.

[0092] Method of the Utilization of Gene

[0093] Probes for detecting SRCL-P1 gene can be specified on the basisof the nucleotide sequence set out in either SEQ ID NO: 1 or 3.Alternatively, primers can be specified for the amplification of DNA orRNA including such a nucleotide sequence. To specify a probe or a primerbased on a given sequence is ordinarily carried out by those skilled inthis art. An oligonucleotide having a specified nucleotide sequence canbe obtained through chemical synthesis. When a suitable label is addedto the oligonucleotide, it can be utilized for hybridization assay inseveral formats. Alternatively, it can be also utilized in reactions forsynthesis of nucleic acids such as PCR. The oligonucleotide that isutilized as a primer is of at least 10 bases in length, and preferablyof 15 to 50 bases in length. It is desirable that the oligonucleotideused as a probe be of from 100 bases to its full length. Further, sucholigonucleotides can be also used for the diagnosis of diseases causedby mutation of an SRCL-P1 gene because they can be used for detectinggenetic mutation encoding SRCL-P1 protein and for detecting SNP. Theyare expected to be available for the diagnosis of a variety of diseasesincluding for example, arteriosclerosis, diabetic complications andAlzheimer's disease, hyper β-lipoproteinemia, hypercholesterolemia,hypertriglyceridemia, hypo α-lipoproteinemia, transplantation,atherectomy, post angiogenic restenosis, bacterial infections, and thelike. In addition, they are also useful for gene therapy whereby SRCL-P1gene is introduced into a living body to allow the expression thereof.

[0094] Moreover, it is also possible to obtain a promoter region and anenhancer region of the SRCL-P1 gene that is present in a genome, basedon a cDNA nucleotide sequence of SRCL-P1 provided by the presentinvention. In particular, these control regions can be obtained bysimilar methods to those disclosed in Japanese unexamined patentpublication No. 6-181767; J. Immunol., 155, 2477, 1995; Proc. Natl.Acad. Sci, USA., 92, 3561, 1995, and the like. Promoter region referredto herein means a DNA region which controls the expression of a genethat exists upstream of a transcription initiation site. Enhancer regionherein means a DNA region that enhances the expression of a gene thatexists in an intron, a 5′-untranslated region, or a 3′-untranslatedregion.

[0095] Method of the Utilization of Protein

[0096] SRCL-P1s proteins of the present invention can be utilized in theelucidation of functions of macrophage and fundamental immunity, theelucidation of mechanisms of development of various types of diseasesincluding for example, arteriosclerosis, diabetic complications andAlzheimer's disease, hyper β-lipoproteinemia, hypercholesterolemia,hypertriglyceridemia, hypo α-lipoproteinemia, transplantation,atherectomy, post angiogenic restenosis and the like, as well asdiagnostic, prophylactic, therapeutic methods thereof, and in thedevelopment of reagents and drugs for the same. Furthermore, they can beused as an antigen for producing antibodies to SRCL-P1s. Additionally,they can be utilized in the screening method of agonists or antagonists.

[0097] Agonist and Antagonist

[0098] The present invention also relates to agonists which stimulatethe activity or the activation of SRCL-P1 of the present invention. Inaddition, the present invention also relates to antagonists whichinhibit the activity or the activation of SRCL-P1 of the presentinvention. For screening the antagonist, a competitive experimentalsystem can be used, for example, in which OxLDL or an antibody, and acandidate inhibitor are subjected to the interaction with cellsexpressing SRCL-P1 protein thereby allowing the candidate inhibitor toscreen based on the binding ratio of OxLDL. Otherwise, conventionallyknown methods may also be carried out to effect the screening. Further,the antagonists also include antisense nucleic acids that inhibit theexpression of SRCL-P1 gene. Included in the examples of the othermethods for the screening may be methods in which a change inextracellular pH is measured, which is caused by the activation of areceptor (Science, 246, 181-296, 1989).

[0099] The antagonist thus screened can be also utilized as a drug forthe treatment, which may include therapy, prophylaxis and the like, ofpathological states involving in the accumulation of oxidized LDL, or inthe binding of AGE to cells. The screening method comprises the steps ofcomparing the amount of binding between SRCL-P1 of the presentinvention, and oxidized LDL or AGE in the presence and absence of acandidate drug; and identifying the candidate drug for treating theintended pathological state by an inhibitory ability of the candidatedrug to the binding therebetween.

[0100] Transgenic Non-Human Animal

[0101] The present invention relates to transgenic non-human animalshaving an altered expression level of SRCL-P1 gene. SRCL-P1 gene hereinincludes cDNA, genomic DNA or synthesized DNA encoding hSRCL-P14 orSRCL-P1. For expression of a gene, any one of the steps of transcriptionand translation should be comprised. The transgenic non-human animalsaccording to the present invention are useful for the investigation offunctions or expression mechanisms of SRCL-P1, elucidation of mechanismsof diseases that are anticipated to be involved in SRCL-P1, developmentof diseased animal models for use in screening and safety tests ofpharmaceutical products.

[0102] In the present invention, the gene can be artificially modifiedto increase or decrease the expression level in comparison with thenative expression level of the gene by introducing mutation such asdeletion, substitution, addition and/or insertion into a part of somekey sites (enhancer, promoter, intron and the like) that regulate theexpression of the gene to be proper. The introduction of the mutationcan be carried out by known methods to obtain a transgenic animal.

[0103] Transgenic animals in their narrow means refer to animals havinggerm cells into which a foreign gene was artificially introduced by agenetic recombination technique. In their broader means, they include:antisense transgenic animals having a particular gene of which functionwas suppressed using an antisense RNA; knockout animals having aparticular gene knocked out using embryonic stem cells (ES cell); andanimals having point mutation of DNA introduced, all of which areanimals having a chromosome with a foreign gene being stably introducedat an early stage of the development of the individual, and having agenotype that can be transmitted to the progeny thereof.

[0104] Transgenic animals referred to herein should be comprehended intheir broader means including all vertebrates other than human. Thetransgenic animals according to the present invention are useful for theinvestigation of functions or expression mechanisms of SRCL-P1,elucidation of mechanisms of diseases that are involved in cellsexpressed in human, development of diseased animal models for use inscreening and safety tests of pharmaceutical products.

[0105] Method for producing a transgenic mouse may include: a method inwhich a gene is directly injected into a nucleus of an ovum in aanterior nucleus phase with a micropipette under a phase contrastmicroscope (microinjection technique, U.S. Pat. No. 4,873,191); a methodin which embryonic stem cells (ES cells) are used. Alternatively, amethod in which a gene is introduced into a retrovirus vector or anadenovirus vector followed by infection into an ovum; a sperm vectortechnique in which a gene is introduced into an ovum via a sperm; andthe like have been developed.

[0106] The sperm vector technique is a genetic recombinant method inwhich a foreign gene is attached to a sperm, or a foreign gene isintroduced into a sperm cell with an electroporation technique, and thenthe foreign gene is introduced into an ovum by fertilizing the ovum (M.Lavitranoet et al., Cell, 57, 717, 1989). Alternatively, site directedgenetic recombination in vivo may be also employed by a cre/locPrecombinase system of bacteriophage P1, a FLP recombinase system ofSaccharomyces cerevisiae, or the like. Additionally, a method has beenalso reported in which a transgene of a desired protein is introducedinto a non-human animal using retrovirus.

[0107] Method for the production of a transgenic animal with amicroinjection technique is carried out as described below, for example.

[0108] First, a transgene is required, which is substantiallyconstituted from a promoter involved in expression control, a geneencoding a specified protein, and a poly(A) signal. The manner of theexpression and/or the expression level of a specified molecule may beaffected by the promoter activity. In addition, because transgenicanimals are different among the produced lineages in respect to thenumber of the copies of the introduced transgene, or the introduced sitein the chromosome, the manner of the expression and/or the expressionlevel must be confirmed-for each of the lineages. Since it has beenelucidated that the expression level is altered depending on theuntranslated region or splicing, an intron sequence to be spliced at apreceding site of poly (A) signal may be previously introduced. It isimportant to use a gene, which is introduced into a fertilized ovum, hasas high purity as possible. The animal to be used may include mice foruse in collecting fertilized ova (5-6 weeks old), male mice for use inmating, female pseudopregnant mice, vas deferens ligated male mice, andthe like.

[0109] In order to efficiently obtain the fertilized ova, gonadotropinor the like may be used for inducing the ovulation. The fertilized ovaare harvested, and thereafter, a gene in an injection pipette isintroduced into a male pronucleus of the ovum by a microinjectiontechnique. An animal (a pseudopregnant mouse or the like) for use inrepositioning the injected ova to an oviduct is provided, to which 10-15ova are transplanted per one animal. Thereafter, the born mouse can beexamined for the introduction of the transgene by: extracting genomicDNA from the end portion of the tail; and detecting the transgene by aSouthern method or a PCR technique, alternatively by a positive cloningtechnique where a marker gene is inserted which is activated upon onlythe occurrence of homologous recombination. Moreover, in order toascertain the expression of the transgene, a transcription productderived from the transgene is detected by a Northern method or a RT-PCRtechnique. Alternatively, a western blotting method may be carried outwith a specific antibody to the protein or a fragment thereof.

[0110] Knockout Mouse

[0111] The knockout mouse according to the present invention is one thatwas treated in a manner to deprive the function of SRCL-P1 gene.Knockout mouse refers to a transgenic mouse in which an arbitrary geneis destroyed by a homologous recombination technique to impair thecorresponding function. The knockout mouse can be produced by homologousrecombination using ES cells, followed by the selection of the embryonicstem cell having one of the allelic gene altered/destroyed. A chimericmouse, which carry cells derived from the embryonic stem cells and cellsderived from the embryo being mixed, may be obtained by, for example,injecting the embryonic stem cell that had been genetically engineeredat blastocyst stage or morulae stage of the fertilized ovum. When thischimeric mouse (chimera refers to a single individual built-up withsomatic cells on the basis of more than two fertilized ova) is crossbredwith a normal mouse, a heterozygotic mouse can be produced having one ofthe allelic gene is entirely altered/destroyed. Further, a homozygoticmouse can be produced by crossbreeding heterozygotic mice each other.

[0112] Homologous recombination refers to the recombination that iscaused by a mechanism of genetic recombination between two genes havingidentical or extremely similar nucleotide sequences. For the selectionof cells with the homologous recombination, PCR can be employed. PCRreaction, in which primers corresponding to a part of the inserted geneand a part of the region expected to be inserted are used, may becarried out to reveal the homologous recombination occurring in cellsthat could yield the amplification products. Also, when the homologousrecombination is caused to a gene expressed in embryonic stem cells, thegene to be introduced may be joined to a neomycin resistant gene toallow the selection after the introduction into cells by making themresistant to neomycin. Accordingly, known methods and the modifiedmethods thereof can be employed to enable the easy selection.

[0113] Method for Producing Antibodies

[0114] The present invention further provides antibodies that recognizeSRCL-P1 or fragments thereof. The antibodies in accordance with thepresent invention include for example, the antibodies to a proteincomprising an amino acid sequence set out in SEQ ID NO: 2 or 4, or afragment thereof. The antibodies (e.g., polyclonal antibodies,monoclonal antibodies, peptide antibodies) or antisera to SRCL-P1 or afragment thereof can be produced using SRCL-P1 or a fragment thereof ofthe present invention as an antigen according to any method forproducing the antibodies or antisera which is known per se. Inparticular, the antibodies that can control the function of SRCL-P1(e.g., antibodies that recognize CRD, a collagen like domain and anα-helical coiled coil domain or the like) are useful for pharmaceuticalproducts containing the antibody.

[0115] SRCL-P1 or a fragment thereof according to the present inventionmay be administered neat or with a diluent or a carrier to awarm-blooded animal at a site that enables the production of theantibody upon the administration. In order to facilitate the productionof antibodies upon the administration, complete Freund's adjuvant orincomplete Freund's adjuvant may be administered. The administration maybe usually conducted once per 1 to 6 weeks, and two to ten times intotal. The warm-blooded animal used may include for example, monkey,rabbit, dog, guinea pig, mouse, rat, sheep, goat, chicken, and the like.Among these, mouse and rat may be preferably used. Rat that may bepreferably used includes Wistar and SD strain rat, and mouse that may bepreferably used includes BALB/c, C57BL/6 and ICR strain mouse and thelike.

[0116] Upon the production of cells that produce a monoclonal antibody,an individual with the antibody titer that can be recognized therein isselected from the warm-blooded animals e.g., mice that had beenimmunized with an antigen. On two to five days after final immunization,spleen or lymph node is collected, and the antibody producing cellscontained therein are subjected to the fusion with myeloma cells toeffect the preparation of monoclonal antibodies producing cells. Thedetermination of the antibody titer in the antiserum may be carried outfor example, by subjecting a labeled SRCL-P1 described below to areaction with the antiserum, and measuring the activity of the labelbound to the antibody. The fusion operation can be performed inaccordance with a known technique for example, the method of Köhler andMilstein (Nature, 256, 495, 1975) and the modified method thereof (J.Immunol. Method, 39, 285, 1980; Eur. J. Biochem., 118, 437, 1981;Nature, 285, 446, 1980). Examples of the fusion accelerating agent mayinclude polyethylene glycol (PEG), Sendai virus and the like, andpolyethylene glycol may be preferably used. In addition, lectin,poly-L-lysine or DMSO may be added ad libitum to raise the efficiency ofthe fusion.

[0117] Examples of the myeloma cell include X-63Ag8, NS-1, P3U1, SP2/0,AP-1 and the like, and SP2/0 may be preferably used. The ratio ofantibody producing cell (spleen cell) number to myeloma cell numberpreferably used is 1:20-20:1. PEG (preferably, PEG1000-PEG6000) is addedat approximately 10-80%. The fusion mixture is incubated at 20-40° C.,preferably at 30-37° C. for 1-10 min. Such a condition enables efficientcell fusion. Screening of the hybridoma that produces anti-SRCL-P1antibody may be performed by using various methods, which include forexample, a method in which a supernatant of hybridoma culture is addedto a solid phase (e.g., a microplate) absorbed with SRCL-P1 antigendirectly or with a carrier, and then an anti-immunoglobulin antibody(when the cell used for the cell fusion was derived from a mouse,anti-mouse immunoglobulin antibody may be used) that was labeled with aradioactive substance, enzyme or the like, or protein A is added theretothereby detecting the anti-SRCL-P1 antibody bound to the solid phase; ora method in which a supernatant of hybridoma culture is added to a solidphase absorbed with anti-immunoglobulin antibody or protein A, and thenSRCL-P1 labeled with a radioactive substance, enzyme or the like isadded thereto thereby detecting the anti-SRCL-P1 monoclonal antibodybound to the solid phase.

[0118] Selection and cloning of the anti-SRCL-P1 antibody can be carriedout by known methods per se, or the modified methods thereof. Usually,the method is carried out in a medium for animal cells added with HAT(hypoxanthine, aminopterin, thymidine). The medium for use in theselection, cloning and growing may be any one of the media in whichhybridoma can grow. For example, RPMI medium containing 1-20%,preferably 10-20% of fetal bovine serum, GIT medium containing 1-10% offetal bovine serum, or serum free medium for hybridoma culture, and thelike. The temperature of the culture may be preferably about 37° C. Theculture period may be usually five days to three weeks, preferably oneweek to two weeks. The culture is usually conducted in the presence of5% carbon dioxide gas. The antibody titer of the supernatant of thehybridoma culture can be measured in a similar manner to the measurementof the antibody titer of anti-SRCL-P1 antibody in an antiserum asdescribed above. In other words, a radioimmunoassay (RIA) technique, anenzyme linked immunosorbent assay (ELISA) technique, a FIA (fluorescentimmunoassay) technique, a plaque measurement technique, an agglutinationreaction technique and the like may be employed as the measurementmethod, however, the ELISA technique as described below is preferred.

[0119] The screening by an ELISA technique can be carried out inaccordance with the following procedure. A protein, which was preparedby a similar method to that for the immunoantigen, is immobilized on thesurface of each well of an ELISA plate. Next, BSA, MSA, OVA, KLH,gelatin or skimmed milk or the like is immobilized for the purpose ofpreventing non-specific adsorption. To each well of this plate addedwith a supernatant solution of the hybridoma culture, followed byallowing the immunoreaction by standing for a predetermined time. Eachwell is washed using a washing solution such as PBS or the likeSurfactant may be preferably added to this washing solution. Anenzyme-labeled secondary antibody is added, and the mixture is allowedto stand for a predetermined time. The enzyme for labeling which can beused includes β-galactosidase, alkaline phosphatase, peroxidase and thelike. After the washes with the same washing solution, enzyme reactionis effected through adding a substrate solution of the labeled enzymethat was employed. When the desired antibody is present in the addedsupernatant solution of the hybridoma culture, the enzyme reactionproceeds to change the color of the substrate solution.

[0120] Cloning can be usually carried out by known methods per se, suchas a semisolid agar technique, a limiting dilution technique or thelike. Specifically, after the well in which the desired antibody isproduced is confirmed by the method described above, a single clone isobtained through conducting the cloning. The method for cloning mayinvolve a limiting dilution technique or the like, in which hybridomacells are diluted so that one colony per one well of a culture plate isformed, and thereafter the culture is conducted. Cloning by a limitingdilution technique may be performed through the use of feeder cells inorder to elevate the colony formation ability, otherwise, a cell growthfactor such as interleukin 6 may be added thereto. Alternatively, FACSand single cell manipulation techniques can be employed for the cloning.The cloned hybridoma is cultured preferably in a serum free medium, andan appropriate amount of the antibody is added to the supernatantthereof. Thus resulting single hybridoma may be subjected to a largescale culture using a flask or a cell culture equipment, or may becultured in the peritoneal cavity of an animal (J. Immunol. Meth., 53,313, 1982) to give a monoclonal antibody. When the culture is conductedin a flask, a medium for cell culture (IMDM, DMEM, RPMI 1640, MEM andthe like) containing 0-20% of FCS can be used. When the culture isconducted in the peritoneal cavity of an animal, an animal of the samespecies, and the same strain as the animal from which myeloma cellsderived that were used for the cell fusion; otherwise an athymic nudemouse may be preferably used. Hybridoma is transplanted after mineraloil such as pristine or the like is previously administered to theanimal. Ascites containing the monoclonal antibody can be obtained afterone to two weeks passed, when the myeloma cells enough proliferate.

[0121] The monoclonal antibody of the present invention can be obtainedas the antibody, which does not cross-react with other proteins, byselecting one which recognizes an epitope specific for SRCL-P1. Ingeneral, an epitope, which is presented by serial amino acid residues ofat least more that or equal to five, preferably 7 to 20 amino acidsamong the amino acid sequence constituting the protein, is referred toas an epitope inherent in the protein. Therefore, the monoclonalantibody that recognizes an epitope constituted from a peptide having anamino acid sequence, which were selected from the amino acid set out inany of SEQ ID NO: 2 and 4, consisting of at least five serial amino acidresidues may be identified as the monoclonal antibody specific tohSRCL-P1 or mSRCL-P1 according to the present invention. When an aminoacid sequence is chosen which is conserved among the amino acid sequenceset out in SEQ ID NO: 2 and 4, an epitope common to SRCL-P1 can beselected. Alternatively, a monoclonal antibody can be selected whichenables the discrimination of each protein, with a region including anamino acid sequence specific for each of the sequences.

[0122] The separation and purification of anti-SRCL-P1 monoclonalantibody can be carried out according to the separation and purificationmethod of an immunoglobulin similarly to the usual separation andpurification method of the polyclonal antibodies. Known purificationmethod which can be adopted may include for example, a saltprecipitation technique, an alcohol precipitation technique, anisoelectric point precipitation technique, an electrophoretic technique,an ammonium sulfate precipitation technique, an adsorption/desorptiontechnique by an ion exchanger (e.g., DEAE), an ultracentrifugationtechnique, a gel filtration technique, and a specific purificationtechnique in which an antibody alone is collected by an antigen-boundsolid phase or an active adsorbent such as protein A or protein G, orthe like, followed by dissociation of the binding to give the antibody.For the purpose of preventing the formation of aggregates, or thedecrease in the antibody titer in the purification step, for example,human serum albumin may be added at a concentration of 0.05-2%.Otherwise, amino acids such as glycine, α-alanine and the like, inparticular, basic amino acid such as lysine, arginine, histidine and thelike, saccharides such as glucose, mannitol and the like, salts such assodium chloride may be also added. In the case of IgM, which is known tobe liable to agglutinate, it may be treated with β-propionolactone andacetic anhydride.

[0123] The polyclonal antibody according to the present invention can beproduced by known methods per se, or the modified methods thereof. Forexample, to produce a polyclonal antibody, an immunoantigen (a proteinantigen) itself or a complex, which was formed with the immunoantigenand a carrier protein, is used for the immunization of a warm-bloodedanimal in a similar manner to the method for producing the monoclonalantibody described above, followed by collecting the preparationcontaining the antibody to the protein of the present invention or afragment thereof from the warm-blooded animal, and then the antibody-ispurified/isolated. In respect to the complex of an immunoantigen and acarrier protein for use in the immunization of the warm-blooded animal,the kind of the carrier protein and the mixing ratio of the carrier andhapten may be optionally determined as long as the antibody can beefficiently produced to the hapten subjected to the immunization aftercrosslinking with the carrier. Thus, any kind of the carrier protein maybe crosslinked at any ratio, however, the method in which about 0.1-20,preferably about 1-5 of bovine serum albumin, bovine thyroglobulin,hemocyanin or the like, for example, is coupled with 1 of hapten byweight may be used. In addition, various condensing agents may be usedfor the coupling of the hapten and carrier, which may includeglutaraldehyde and carbodiimide, and active ester reagents containingmaleimide active ester, thiol group, dithiopyridyl group and the like.The condensation product is administered neat or with a carrier or adiluent to a warm-blooded animal at a site that enables the productionof the antibody upon the administration. In order to facilitate theproduction of antibodies upon the administration, complete Freund'sadjuvant or incomplete Freund's adjuvant may be administered. Theadministration may be usually conducted once per 2 to 6 weeks, and threeto ten times in total. Polyclonal antibodies can be collected from theblood, ascites and the like, and preferably from the blood, of thewarm-blooded animal immunized by a method as described above. Themeasurement of the antibody titer in antiserum can be carried out in asimilar manner to the measurement of the antibody titer of the antiserumas described above. The separation and purification of the polyclonalantibody can be carried out according to the separation and purificationmethod of an immunoglobulin similarly to the separation and purificationmethod of a monoclonal antibody described above.

[0124] Method of the Utilization of Antibody

[0125] Monoclonal antibodies and polyclonal antibodies to SRCL-P1 or afragment thereof can be utilized in diagnosis and therapy of thediseases relating to the cells that are expressing SRCL-P1. SRCL-P1 or afragment thereof can be measured using these antibodies, on the basis ofthe immunological binding with SRCL-P1 or the fragment thereof accordingto the present invention. Specifically, the method for measuring SRCL-P1or a fragment thereof using such an antibody may include for example,sandwich techniques in which a sandwich complex is detected which wasproduced by subjecting SRCL-P1 or a fragment thereof to a reaction withan antibody coupled to an insoluble support and a labeled antibody; orcompetitive techniques in which SRCL-P1 or a fragment thereof in asample is measured by subjecting labeled SRCL-P1, and SRCL-P1 or afragment thereof in a sample to a competitive reaction with the antibodyfollowed by the measurement of SRCL-P1 or a fragment thereof in a samplefrom the amount of the labeled antigen that reacted with the antibody.

[0126] Upon the measurement of SRCL-P1 or a fragment thereof by thesandwich technique, two-step methods in which SRCL-P1 or a fragmentthereof is first subjected to a reaction with an immobilized antibody;thereafter, unreacted materials are completely removed by washes; andthen a labeled antibody is added thereto to have the immobilizedantibody—SRCL-P1 labeled antibody formed, alternatively, one-stepmethods in which an immobilized antibody, a labeled antibody and SRCL-P1or a fragment thereof are mixed concurrently.

[0127] Insoluble support for use in the measurement include for example,synthetic resin such as polystyrene, polyethylene, polypropylene,polyvinyl chloride, polyester, polyacrylic acid ester, nylon,polyacetal, fluorine-contained resin and the like; polysaccharides suchas cellulose, agarose and the like; glasses; metals; and the like. Theinsoluble support may be in a variety of forms, and for example,tray-like, spherical, fibrous, cylindrical, discal, vessel-like,cell-like, tubular, and the like may be adopted. The support onto whichthe antibody had been adsorbed may be stored in cold, if necessary, inthe presence of an antiseptic agent such as sodium azide and the like.

[0128] For the immobilization of the antibody, known chemical couplingmethods or physical adsorption methods may be adopted. Chemical couplingmethod includes for example, methods in which glutaraldehyde is used;maleimide methods in which N-succinimidyl-4-(N-maleimidemethyl)cyclohexane-1-carboxylate and N-succinimidyl-2-maleimide acetate and thelike are used; carbodiimide methods in which1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride and thelike is used. Other method includesmaleimidebenzoyl-N-hydroxysuccinimide ester methods,N-succimidyl-3-(2-pyridylthio) propionic acid methods, bisdiazobenzidinemethods, dipalmityl lysine methods. Alternatively, a complex that hadbeen formed previously by subjecting the substance to be detected to areaction with two kinds of antibodies of which epitopes are differentcan be captured by the third antibody to the antibody, which had beenimmobilized in a similar manner to those described above.

[0129] The material to be used for labeling may include enzyme,fluorescent materials, luminescence materials, radioactive materials,metal chelates and the like. Examples of enzyme may include peroxidase,alkaline phosphatase, β-D-galactosidase, malate dehydrogenase,staphylococcus nuclease, delta-5-steroid isomerase, α-glycerolphosphatedehydrogenase, triose phosphate isomerase, horseradish peroxidase,asparaginase, glucose oxidase, ribonuclease, urease, catalase,glucose-6-phosphate dehydrogenase, glucoamylase, acetylcholine esteraseand the like. Fluorescent materials may include for example, fluoresceinisothiocyanate, phycobilin protein, rhodamine, phycoerythrin,phycocyanin, allophycocyanin, orthophthalic aldehyde and the like.Luminescence materials may include isoluminol, lucigenin, luminol,aromatic acridinium esters, imidazole, acridinium salts and modifiedesters thereof, luciferin, luciferase, aequorin and the like.Radioactive materials may include ¹²⁵I, ¹²⁷I, ¹³¹I, ¹⁴C, ³H, ³²P, ³⁵Sand the like. These materials are not limited thereto as long as thematerial can be used in immunological determination methods. Inaddition, low molecular weight hapten such as biotin, dinitrophenyl,pyridoxal or fluorescamine may be conjugated to the antibody.Preferably, horseradish peroxidase may be used as a labeling enzyme.This enzyme can react with many kinds of substrates, which can bereadily conjugated to the antibody by a periodic acid method.

[0130] When an enzyme is used as a labeling agent, a substrate formeasuring its activity, and a color-developing agent as needed may beemployed. When peroxidase is used as an enzyme, H₂O₂ may be used as asubstrate solution, and 2,2′-azino-di-[3-ethylbenzthiazolin sulfonate]ammonium (ABTS), 5-aminosalicylic acid, orthophenylenediamine,4-aminoantipyrine, 3,3′,5,5′-tetramethylbenzidine or the like may beused as a color-developing agent. When alkaline phosphatase is employedas an enzyme, orthophenylphosphate, paranitrophenylphosphate or the likemay be used as a substrate. Alternatively, when β-D-galactosidase isused as an enzyme, fluorescein-di-(β-D-galactopyranoside),4-methyl-umbelliferyl-D-galactopyranoside, or the like may be used as asubstrate. The present invention also involves kit products including amonoclonal antibody, a polyclonal body described above, and reagents.

[0131] Available crosslinking agents includeN,N′-orthophenylenedimaleimide, 4-(N-maleimidemethyl) cyclohexanoylN-succinimide ester, 6-maleimidehexanoyl N-succinimide ester,4,4′-dithiopyridine, and other known crosslinking agents. The reactionof such a crosslinking agent with the enzyme and the antibody may beconducted in accordance with known methods depending upon the propertiesof the respective crosslinking agents. Additionally, the antibodies tobe used may be any fragments of the antibodies for example, Fab′, Fab,F(ab′)₂ depending on the condition. Furthermore, enzymatically labeledantibodies may be prepared by using a similar method to any one of thosefor polyclonal antibodies and monoclonal antibodies. When theenzymatically labeled antibody that was obtained by using theaforementioned crosslinking agent is purified by any known methods suchas affinity chromatography or the like, more sensitive immunologicaldetermination system can be achieved. The enzymatically labeledantibody, which was purified in such a manner, is stored in a cold anddark place after adding thimerosal, glycerol or the like as astabilizer, alternatively, after being lyophilized.

[0132] The subject sample for the measurement may be a sample containingSRCL-P1, which may include body fluids such as plasma, serum, blood,urine, tissue fluid, cerebrospinal fluid and the like, various types ofcells, tissues, and the like.

[0133] Method for Producing Humanized Antibody

[0134] It is ethically impermissible to produce antibodies by immunizinghuman with an optional antigen. Further, when a mouse monoclonalantibody is administered to a human body, there is a risk of theoccurrence of a variety of adverse effects, because the antibody is aheterogeneous protein to human. Therefore, an antibody with loweredantigenicity to human is preferred when the antibody is administered tohuman.

[0135] Method for the production of human monoclonal antibodies involvestransformation techniques with Epstein-Barr virus (EBV), and fusiontechniques in which thus transformed cells and parent cells are fused;methodes in which a chimeric antibody or a humanized antibody isproduced using genetic engineering techniques; and the like in additionto cell fusion techniques. Chimeric antibody refers to an antibody thatwas produced by linking immunoglobulin gene fragments from heterogeneousanimals. Humanized antibody refers to an antibody having a substitutedprimary structure in part other than a complementarity determiningregion (CDR) of H chain and L chain with the corresponding primarystructure of a human antibody through introducing the alteration to amouse antibody or the like that is heterogeneous to human.

[0136] For the production of a chimeric antibody, a mouse is immunizedfirst, and an antibody variable region (V region) that binds to anantigen is excised from a gene of the mouse monoclonal antibody.Thereafter, the V region is linked to a gene of an antibody constantregion (C region) derived from human myeloma to give a chimeric gene.Upon expression of this chimeric gene in a host cell, human-mousemonoclonal antibody can be produced. Because chimeric antibodies areless antigenic to human, they can be utilized as a monoclonal antibodyfor therapeutic use to be administered into a human body, or for use indiagnostic imaging. Known techniques relevant to chimeric antibodiesinvolve Japanese patent unexamined publication No. Hei 05-304989,Japanese patent unexamined publication No. Hei 04-330295, WO9106649,Japanese patent unexamined publication No. Sho 63-036786, Japanesepatent examined publication No. Hei 06-98021, and the like.

[0137] Moreover, humanized antibodies were recently developed, which areappreciated as being more useful than chimeric antibodies. Humanizedantibody refers to an antibody that is humanized as a whole moleculeexcept for CDR of an antibody molecule by grafting only a sequence of agene for an antigen-binding site (CDR: complementarity determiningregion) of an antibody molecule into a gene of a human antibody (CDRgrafting). This antibody is appreciated as being safer with lessantigenicity than the human -mouse chimeric antibody because it has lesspart derived from a mouse antibody. When SHM-D 33 strain (ATCC CRL 1668)or RF-S1 strain, both of which being human/mouse heteromyeloma, is usedas a parent cell for producing a human monoclonal antibody, high fusionefficiency can be achieved that is equivalent to mouse parent cells.Hybridoma that was obtained using these parent cells can be clonedwithout feeder cells, and it can produce IgG type antibody in acomparatively stable manner at a large amount. For the culture of theparent cells, ERDF medium supplemented with 15% FCS may be used,although other operation may be similarly carried out to the operationfor the murine cells. Additionally, in order to produce an IgG typehuman monoclonal antibody, human lymphocytes collected from peripheralblood may be preferably employed, which were sufficiently sensitizedwith an antigen. When it is difficult to obtain sufficiently sensitizedlymphocytes, sensitization with an antigen may be also conducted invitro. In Japan, clinical trials have been currently carried out forhumanized antibodies to adult T cell leukemia. In respect to theproduction of human antibodies and the related art, for example,reference should be made to those disclosed in Genentech Inc., USA(WO9222653, WO9845332, WO9404679, WO9837200, WO9404679) and CelltechInc., England (WO9429451, WO9429351, WO9413805, WO9306231, WO9201059,WO9116927, WO9116928, WO9109967, WO8901974, WO8901783), and the like.

[0138] Using the methods and the like described above, the antibodiesaccording to the present invention can be humanized, and such antibodieswould be extremely useful upon the administration to human.

[0139] Composition

[0140] The SRCL-P1 polynucleotides or proteins and antibody substances,and antagonists and the like of SRCL-P1 are possibly utilized indiagnostic, prophylactic and therapeutic methods, and for thedevelopment of reagents and drugs for various types of diseases involvedin the accumulation of oxidized LDL (modified LDL) including forexample, arteriosclerosis and the like, as well as disorders involved inthe binding of AGE to cells such as glomerulosclerosis and the like,diabetic complications and AD, hyper β-lipoproteinemia,hypercholesterolemia, hypertriglyceridemia, hypo α-lipoproteinemia,transplantation, atherectomy, and post angiogenic restenosis, bacterialinfections and the like. Further, the ingredient can be combined orblended with known medical drugs. For example, the ingredient can becombined or blended with therapeutic drugs of atherosclerosis, e.g.,ACAT inhibitors, HMG-CoA reductase inhibitors, lipid regulants, bileacid regulants.

[0141] Pharmaceutical composition according to the present invention maycomprise SRCL-P1 polynucleotides or proteins, substances that stimulateor inhibit the activity or activation of SRCL-P1 protein, substancesincluding antibodies to SRCL-P1 protein and the like (hereinafter,referred to as “SRCL-P1 related substance”). The SRCL-P1 relatedsubstances can be used neat, or after subjecting to several kinds oftreatment such as dilution in water and the like, and they may also beused after blending in pharmaceutical products, quasi drugs and thelike. In these cases, the amount of the substance to be blended may bedetermined ad libitum. When the substance is formulated for the systemicadministration, 0.001-50% by weight, in particular, 0.01-10% by weightis permissible. When the amount is less than 0.001%, sufficient actionof lacrimation may not be enabled. When the amount is greater than 50%,properties such as stability, flavor and the like of the compositionitself may be deteriorated.

[0142] The route of administration can be optionally selected from theadministration via mucosa, transdermal administration, intramuscularadministration, subcutaneous administration, endorectal administration,topical ocular administration, and the like, in addition to oraladministration and intravenous administration described above.

[0143] The SRCL-P1 realated substance according to the present inventionmay be included in the formulation as a salt. Pharmaceuticallyacceptable salts include for example, salts with base such as inorganicbase, organic base and the like; acid addition salts such as those ofinorganic acid, organic acid, basic or acidic amino acid. Inorganicbases include for example, alkaline metal such as sodium, potassium andthe like; alkaline earth metal such as calcium, magnesium and the like;aluminum, ammonium and the like. Organic bases include for example,primary amines such as ethanolamine and the like; secondary amines suchas diethylamine, diethanolamine, dicyclohexylamine,N,N′-dibenzylethylenediamine and the like; tertiary amines such astrimethylamine, triethylamine, pyridine, picoline, triethanolamine andthe like. Inorganic acids include for example, hydrochloric acid,hydrobromic acid, nitric acid, sulfuric acid, phosphoric acid and thelike. Organic acids include for example, formic acid, acetic acid,lactic acid, trifluoroacetic acid, fumaric acid, oxalic acid, tartaricacid, maleic acid, benzoic acid, citric acid, succinic acid, malic acid,methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid,p-toluenesulfonic acid and the like. Basic amino acids include forexample, arginine, lysine, ornithine and the like. Acidic amino acidsinclude for example, aspartic acid, glutamic acid and the like.

[0144] Examples of dosage forms for use in oral administration includepowdered formulations, granulated formulations, encapsulatedformulations, pills, tablets, elixirs, suspensions, emulsions, syrupsand the like, which may be selected ad libitum. In addition, suchformulations may be modified, which may involve release control,stabilization, facilitation of disintegration, blocking ofdisintegration, enteric coating, facilitation of absorption and thelike. Moreover, examples of dosage forms for the intraoral topicaladministration include chewable formulations, sublingual formulations,buccal formulations, lozenges, ointments, plasters, liquid formulationsand the like, which may be selected ad libitum. Further, suchformulations may be modified, which may involve release control,stabilization, facilitation of disintegration, blocking ofdisintegration, enteric coating, facilitation of absorption and thelike.

[0145] Known drug delivery system (DDS) techniques may be applied todosage forms as described above. DDS formulation referred to hereininvolves sustained release formulations, topically applied formulations(lozenges, buccal formulations, sublingual formulations), drugcontrolled release formulations, enteric coated formulations,formulations soluble in stomach and the like, which are formulationsthat are prepared so that most appropriate dosage form is accomplishedtaking into account of the administration route, bioavailability,adverse effect and the like.

[0146] Components for DDS essentially comprise a drug, a drug releasemodule, a coating and a therapy program. In detail, the drug having ashort half life is preferred, which permits rapid decline of the bloodconcentration particularly upon cessation of the release thereof. Thecoating is preferably nonreactive to the body tissue of the part towhich the drug is administered. In addition, the therapy program ispreferably configured so that the most optimal drug concentration iskept during the predetermined period. The drug release modulesubstantially has a drug storage, a release control part, an energysource, and a release opening or a release surface. All of thesefundamental components are not necessarily required, and thus addition,deletion or the like may be optionally carried out to select the bestmode.

[0147] Examples of materials which can be used for DDS include polymers,cyclodextrin derivatives, lecithin and the like. The polymer may includeinsoluble polymers (silicone, ethylene-vinyl acetate copolymer,ethylene-vinyl alcohol copolymer, ethylcellulose, cellulose acetate andthe like), water soluble polymers and hydroxyl gel-forming polymers(polyacrylamide, polyhydroxyethyl methacrylate cross-linked form,polyacryl cross-linked form, polyvinyl alcohol, polyethyleneoxide, watersoluble cellulose derivatives, cross-linked poloxamer, chitin, chitosanand the like), slow dissolving polymers (ethyl cellulose, a partialester of methylvinyl ether-maleic anhydride copolymer and the like),polymers soluble in stomach (hydroxylpropylmethyl cellulose,hydroxylpropyl cellulose, carmellose sodium, macrogol,polyvinylpyrrolidone, dimethylaminoethyl methacrylate-methylmethacrylate copolymer and the like), enteric polymers(hydroxylpropylmethyl cellulose phthalate, cellulose acetate phthalate,hydroxylpropylmethyl cellulose acetate succinate, carboxymethylethylcellulose, acrylic acid polymers and the like), biodegradable polymers(heat coagulation or cross-linked albumin, cross-linked gelatin,collagen, fibrin, polycyanoacrylate, polyglycolic acid, polylactic acid,poly β-hydroxyacetic acid, polycaprolactone and the like), which can beselected ad libitum on the basis of the dosage form.

[0148] In particular, silicone, ethylene-vinyl acetate copolymer,ethylene-vinyl alcohol copolymer, a partial ester of methylvinylether-maleic anhydride copolymer can be used for the control of drugrelease; cellulose acetate can be used as a material of a osmoticpressure pump; ethyl cellulose, hydroxypropylmethyl cellulose,hydroxypropyl cellulose, methyl cellulose can be used as a material of amembrane of slow dissolving formulations; and polyacryl cross-linkedform can be used as an attaching agent to oral mucosa or ophthalmicmucosa.

[0149] Further, the formulation can be manufactured with adding solvent,excipient, coating agent, base, binding agent, lubricant, disintegrant,solubilizing agent, suspending agent, thickening agent, emulsifyingagent, stabilizing agent, buffering agent, isotonizing agent, soothingagent, preservative agent, flavoring agent, fragrance agent, coloringagent and the like in compliance with its dosage form (known dosagesform such as forms for oral administration, injection, suppository andthe like).

[0150] Although specific examples are respectively illustrated below,these examples should not be construed as limiting the presentinvention.

[0151] [solvent] purified water, water for injection, saline, peanutoil, ethanol, glycerol;

[0152] [excipient] starches, lactose, glucose, sucrose, crystallinecellulose, calcium sulfate, calcium carbonate, talc, titanium oxide,trehalose, xylitol;

[0153] [coating agent] sucrose, gelatin, cellulose acetate phthalate andpolymers as described above;

[0154] [base] vaseline, vegetable oil, macrogol, base for oil in wateremulsion, base for water in oil emulsion;

[0155] [binding agent] natural polymer compounds such as starch andderivatives thereof, cellulose and derivatives thereof, gelatin, sodiumalginate, gum tragacanth, gum arabic, and the like; synthetic polymerssuch as polyvinylpyrrolidone and the like; dextrin, hydroxylpropylstarch;

[0156] [lubricant] stearic acid and salts thereof, talc, waxes, wheatstarch, macrogol, hydrogenated vegetable oil, sucrose fatty acid ester,polyethylene glycol;

[0157] [disintegrant] starch and derivatives thereof, agar, gelatinpowder, sodium bicarbonate, cellulose and derivatives thereof,carmellose calcium, hydroxypropyl starch, carboxymethyl cellulose, andsalts and derivatives thereof, poorly substituted hydroxypropylcellulose;

[0158] [solubilizing agent] cyclodextrin, ethanol, propylene glycol,polyethylene glycol;

[0159] [suspending agent] gum arabic, gum tragacanth, sodium alginate,aluminum monostearate, citric acid, various surfactants;

[0160] [thickening agent] carmellose sodium, polyvinylpyrrolidone,methyl cellulose, hydroxypropylmethyl cellulose, polyvinyl alcohol, gumtragacanth, gum arabic, sodium alginate;

[0161] [emulsifying agent] gum arabic, cholesterol, gum tragacanth,methyl cellulose, various surfactants, lecithin;

[0162] [stabilizing agent] sodium bisulfite, ascorbic acid, tocopherol,chelating agent, inert gas, reducing agent;

[0163] [buffering agent] sodium hydrogenphosphate, sodium acetate, boricacid;

[0164] [isotonizing agent] sodium chloride, glucose;

[0165] [soothing agent] procaine hydrochloride, lidocaine, benzylalcohol;

[0166] [preservative agent] benzoic acid and salts thereof,p-hydroxybenzoic esters, chlorobutanol, inverted soap, benzyl alcohol,phenol, thimerosal;

[0167] [flavoring agent] sucrose, saccharin, glycyrrhiza extract,sorbitol, xylitol, glycerol;

[0168] [fragrance agent] orange peel tincture, rose oil;

[0169] [coloring agent] water soluble edible dye, lake dye.

EXAMPLES

[0170] Novel scavenger receptor according to the present invention isdescribed in more detail by the following non-limiting illustrativeexamples. However, the present invention should not be construed to belimited by the examples.

[0171] Specifically, search on EST database (Example 1); preparation ofprobes for the screening (Example 2); screening of a cDNA library fromhuman placenta (Example 3); base sequencing of novel human scavengerreceptor (Example 4); and obtaining novel mouse scavenger receptor cDNA(Example 5); as well as method for producing a transfectant thattransiently expresses the novel human scavenger receptor (Examples 6 and7); method for producing a transfectant that stably expresses the novelhuman scavenger receptor (Examples 8 and 9); verification of the bindingspecificity of the novel human scavenger receptor (Example 10);demonstration of phagocytic capacity (Example 11); and demonstration ofexpression in vascular endothelial cells (Example 12) are describedbelow, all of which were illustrated.

Example 1 Search on EST Database

[0172] By comparing amino acid sequences of known collecting, i.e.,human MBP, human SP-A and human SP-D (see, FIGS. 2 and 3, wherein,circumscribed amino acid residues denote the part that are recognized tobe homologous), a region highly conserved between the molecules wassearched. Consequently, it was revealed that 27 amino acidscorresponding to from position 220 to position 246 of the human MBPamino acid sequence (FIG. 3, outlined characters, SEQ ID NO: 5) werehighly conserved. Therefore, several consensus sequences in compliancewith this region were produced, and search on EST (Expressed SequenceTags) database was conducted. EST database that was employed contained676750 sequences dated Oct. 11, 1996.

[0173] As a result, several data were obtained for highly homologousamino acid sequence to the 27 amino acids described above. The aminoacid sequences of thus resultant data were searched on GenBank/ESTdatabase, and determined whether they were any of known or unknownsubstance. Consequently, two data (accession number: W72977 and R74387)that exhibit high homology but contain unknown nucleotide sequence couldbe obtained among data that were obtained when the amino acid sequenceset out below was used as a consensus sequence:

[0174]Glu-Lys-Cys-Val-Glu-Met-Tyr-Thr-Asp-Gly-Lys-Trp-Asn-Asp-Arg-Asn-Cys-Leu-Gln-Ser-Arg-Leu-Ala-Ile-Cys-Glu-Phe(SEQ ID NO: 6).

[0175] These data were respectively derived from placenta and fetalheart, which represent a part of the nucleotide sequence of a novelcollectin.

[0176] Accordingly, a clone derived from fetal heart (I.M.A.G.E.Consortium Clone ID 34472) was purchased from ATCC (American TypeCulture Collection) among these, and utilized in the preparation ofprobes for the screening to obtain a novel scavenger receptor below.

Example 2 Preparation of Probes for Screening

[0177] The nucleotide sequence of an insert DNA of the above-describedclone was sequenced with a primer (Pharmacia Co., M13 Universal Primer(SEQ ID NO: 7,5′-fluorescein-cgacgttgtaaaacgacggccagt-3′) and M13Reverse Primer (SEQ ID NO: 8,5′-fluorescein-caggaaacagctatgac-3′)).

[0178] Thus resulting nucleotide sequences was aligned to the amino acidsequence of an open reading flame of a collectin, and then a nucleotidesequence corresponding to the amino acid sequence that can be readtherefrom was extracted. Primers for digoxigenin (DIG) labeled cDNAprobe that correspond to a part of the extracted sequence (Reverseprimer: caatctgatgagaaggtgatg (SEQ ID NO: 9) and Forward primer:acgaggggctggatgggacat (SEQ ID NO: 10)) were produced using AppliedBiosystems Inc., 392A DNA/RNA synthesizer. DIG labeling was conductedusing a PCR DIG probe synthesis kit (Boeringer Mannheim Co., Ltd). Theconstitution of a reaction is as follows: 2 μl (100 ng) of plasmid DNA(clone W72977, 50 ng/μl); 5 μl of 10×buffer; 5 μl of 25 mM MgCl₂; 5 μlof dNTP (PCR labeling mix); 2.5 μl of 20 μM Reverse primer; 5 μl of 20μM Forward primer; 28 μl of H₂O; 0.5 μl of Taq polymerase. PCR reactionwas performed using Atto Co., Ltd., Zymoreactor, with 35 cycles of 92°C. for 1 min, 55° C. for 1 min, and 72° C. for 2 min.

Example 3 Screening of cDNA Library Derived from Human Placenta

[0179] A phage cDNA library derived from human placenta was firstsubjected to the titration as follows. A solution of 0.2 ml ofEscherichia coli Y1090r, which had been cultured in mLB medium (LBmedium (1 g triptone, 0.5 g yeast extract, 0.5 g NaCl/100 ml) containing10 mM MgSO₄ and 0.2% maltose) at 37° C. for 16 hours, and 0.1 ml of cDNAlibrary serially diluted with SM buffer (5.8 g NaCl, 2 g MgSO₄.7H₂O, 25ml of 2 M Tris-HCl (pH 7.5), 5 ml of 2% gelatin/L) were incubated at 37°C. for 15 min. Thereafter, the mixture was added to 2.5 ml of LB-TOPagarose (0.75% agarose/LB medium) to give a homogenous mixture, andplated on 90 mmΦ LB medium plate (Iwaki glass Co., Ltd.) (1.5% agar/LBmedium). The medium was hardened in 15 minutes at room temperature,followed by incubation at 42° C. for 5 hours. After counting the plaquenumber of each plate, the titer of phage of each plate was calculated.As a result, the titer was calculated to be 2.1×10¹⁰ pfu/ml. Thustitrated cDNA library was screened as follows using the probes producedin accordance with Example 2.

[0180] A solution of 0.6 ml of Escherichia coli Y1090r, which had beencultured in mLB medium at 37° C. for 16 hours, and 1×10⁵ pfu of the cDNAlibrary diluted with SM buffer were incubated at 37° C. for 15 min.Thereafter, the mixture was added to 7.5 ml of LB-TOP agarose (0.75%agarose) to give a homogenous mixture. Ten plates of 140 mm² of LBmedium rectangular plate (Nissui Seiyaku Co., Ltd.) to which the mixturewas plated were provided, and hardened in 15 minutes at roomtemperature, followed by incubation at 42° C. for 5 hours. Afterconfirming the plaque formation, transfer to a nylon membrane wasconducted. The transfer was carried out using Nytran 13N (Schleicher andSchuell Co.). A filter of 12.5 cm×9.0 cm was immersed in distilledwater, and moisturized for 10 minutes. Thereafter, the excess moisturewas eliminated on a Whatman 3MM paper, and the filter was placed on theplate with the plaques formed. After leaving to stand for two minutes,the filter was stripped off, and air dried for 10 minutes. Phage DNA wasmodified with 0.2 M NaOH/1.5 M NaCl for 2 min, and neutralized with 0.4M Tris-HCl(pH7.6)/2×SSC for 2 min, followed by washes with 2×SSC for 2min. Thereafter, the phage DNA was fixed on the membrane by ultravioletirradiation using GS GENE LINKER (Bio-Rad Inc.,). Detection of thehybridization and signals was executed as described below. The filterwas moisturized with 2×SSC, and the excess moisture was eliminated witha Whatman 3MM paper. Then the filter was placed into a hybridizationbag, and subjected to prehybridization in a hybridization solution(5×SSC, 1% blocking agent, 0.1% N-lauroylsarcosine, 0.02% SDS) at 68° C.for 1 hour. Subsequently, the hybridization solution was removed fromthe bag, and thereto added a hybridization solution that was prepared togive 10 ng/ml of DIG labeled cDNA probe. Hybridization was carried outat 55° C. for 16 hours. After the hybridization was completed, thefilter was washed with 2×SSC/0.1% SDS solution for 5 min twice at roomtemperature, and with 0.5×SSC/0.1% SDS solution for 15 min twice at 55°C. Next, SDS was removed with DIG buffer I (100 mM Tris-HCl, 150 mM NaCl(pH7.5)) for 1 minute, followed by blocking of the filter with DIGbuffer II(1% blocking agent, DIG buffer I) for 30 min. After the washwith DIG buffer I for 1 min, an antibody reaction was then allowed for30 min through the addition of a solution of 5,000×anti-DIG alkalinephosphatase labeled antibody (Boeringer Mannheim Co., Ltd.) diluted withDIG buffer II. The filter was washed twice with DIG buffer I for 15 minat room temperature. The concentration of Mg²⁺ was elevated by thetreatment with DIG buffer III (100 mM Tris-HCl, 100 mM NaCl (pH 9.5), 50mM MgCl₂) for 3 min, and then a solution of NBT/BCIP (Wako Pure ChemicalIndustries, Ltd.) in DIG buffer III was added to the mixture for thecolor development. Accordingly, ten positive clones were obtained.Plaques corresponding to these clones were excised, and they wererespectively placed in a tube containing 1 ml of SM buffer. Afterstirring for 10 min, the solution was serially diluted with SM buffer.Then, 0.1 ml of thus diluted solution was mixed with a solution of 0.2ml of Escherichia coli Y1090r that had been cultured in mLB medium at37° C. for 16 hours, and the mixture was incubated at 37° C. for 15 min.Thereafter, the mixture was added to 2.5 ml of LB-TOP agarose to give ahomogenous mixture, and plated on 90 mmΦ LB medium plate. Ten platesprepared in this manner were hardened in 15 minutes at room temperature,followed by incubation at 42° C. for 5 hours. Several plaques wereobtained, which were subjected to the secondary screening similarly tothe primary screening.

Example 4 Sequencing of Nucleotide Sequence of Human Novel ScavengerReceptor

[0181] A plaque of a clone, which was deemed to be proper among thepositive clones obtained in the secondary screening, was excised fromthe plate. Thus resulting plaque was placed in a tube containing 200 μlof distilled water. After stirring for 30 min, the solution wascentrifuged at 15,000 rpm for 5 min to give a supernatant.

[0182] Thus resultant supernatant was used as a template to amplify aninsert DNA by PCR using TaKaRa LA PCR Kit Ver.2 (Takara Shuzo Co.,Ltd.). The constitution of the reaction is as follows: 27 μl of thesupernatant, 5 μl of 10×LA PCR buffer II (Mg²⁺ free), 5 μl of 25 mMMgCl₂, 8 μl of dNTP mix, 2.5 μl of 20 μM λgt11 Reverse Primer (SEQ IDNO: 11, 5′-ttgacaccagaccaactggtaatg-3′), 2.5 μl of 20 μM λgt11 ForwardPrimer (SEQ ID NO: 12, 5′-ggtggcgacgactcctggagcccg-3′), 0.5 μl of LA Taqpolymerase, H₂O added to adjust the total volume of 50 μl). The PCRreaction was carried out using Gene Amp PCR system 9600 manufactured byApplied Biosystems Inc., with 30 cycles of at 98° C. for 20 seconds andat 68° C. for 5 minutes. The PCR product was confirmed on 1% agarose gelelectrophoresis, and was purified by excising from the gel. SephaglasBandPrep Kit manufactured by Pharmacia Co., was used for thepurification.

[0183] The DNA fragment excised was incorporated into pCR2.1 vectorincluded in TA cloning kit manufactured by Invitrogen Co,. Therecombinant vector was transformed into TOP10F′ cells included in the TAcloning kit manufactured by Invitrogen Co,. The transformant wascultured in LB medium (100 μg/ml ampicillin), and then three kinds ofplasmid DNA per each clone were extracted by an alkali SDS method.

[0184] Thus resulting DNA was cut with a restriction enzyme that wasenvisaged to be suitable. Each DNA fragment was incorporated into pUC18vector and transformed into XL-1 Blue cells. The transformant wascultured in LB medium (100 μg/ml ampicillin), and then a plasmid wasextracted by an alkali SDS method. Accordingly, the plasmids below wereobtained: a plasmid containing an EcoR I-Hind III fragment or a HindIII-EcoR I fragment from CL-P1-2-1; a plasmid containing an EcoR I-BamHI fragment, a BamH I-Sma I fragment, a Sma I-Hind III fragment, a KpnI-Sau3A I fragment, a Sau3A I-EcoR I fragment, an EcoR I-Kpn I fragmentor an EcoR I-Sma I fragment from CL-P1-3-4; a plasmid containing an EcoRI-BamH I fragment, a BamH I-Sma I fragment, a Sma I-Hind III fragment, aKpn I-Sau3A I fragment, a Sau3A I-EcoR I fragment, an EcoR I-Kpn Ifragment or a Kpn I-EcoR I fragment from CL-P1-3-7. Primers for the usewere: M13 Universal Primer (SEQ ID NO: 7) and M13 Reverse Primer(SEQ IDNO: 8) attached to AutoRead Sequencing Kit (Pharmacia Co.,), and thefollowing primers produced by using a DNA/RNA synthesizer, which werelabeled with FITC (Pharmacia Co., FluorePrime), followed by sequencingof the nucleotide sequence of their entire regions with AutoReadSequencing Kit and A.L.F. Auto Sequencer manufactured by Pharmacia Co.(SEQ ID NO:13) HPP 1: 5′-fluorescein-cgtgaaaatgaatggaagtgg-3′, (SEQ IDNO:14) HPP 2: 5′-fluorescein-ttttatccattgctgttcctc-3′, (SEQ ID NO:15)HPP 3: 5′-fluorescein-ctggcagtccccgaggtccag-3′, (SEQ ID NO:16) HPP 5:5′-fluorescein-gctggtccccccggagagcgt-3′.

[0185] Outline of the sequencing of the nucleotide sequences conductedas above is shown in FIG. 4. FIG. 4(a) represents ORF of acollectin-like structural part of the obtained scavenger receptor, inwhich G-X—Y (wherein G denotes glycine, X and Y may be any one of aminoacid residues) represents a collagen-like domain. In addition, FIG. 4(b)represents each name of the primers described above, the nucleotidesequence that was read by a sequencer, which is shown by arrows, and M13Universal Primer (shown as “U”) and M13 Reverse Primer(shown as “R”).

[0186] Moreover, the nucleotide sequence of 5′ end region including atranscription initiation site of this sequence was determined using Capsite cDNA.

[0187] First PCR was carried out with Cap Site cDNA, Human Liver (NIPPONGENE KK) using 1RC2 Primer (5′-caaggtacgccacagcgtatg-3′ (SEQ ID NO: 17))attached thereto, and TGP1 Primer (5′-tcttcagtttccctaatccc-3′ (SEQ IDNO: 18)) that was synthesized by 392A DNA/RNA synthesizer manufacturedby Applied Biosystems Inc. The reaction mixture contained LA PCR BufferII ( Mg²⁺ free), 2.5 mM MgCl₂, 1 μl of each 200 μM dATP, dCTP, dGTP anddTTP (all of which were manufactured by Takara Shuzo Co., Ltd.), CapSite cDNA Human Liver, 0.5 μM 1RC2 Primer (manufactured by NIPPON GENEKK), and 0.5 μM TGP1 Primer in a total liquid volume of 50 μl. PCR wascarried out with a program involving 35 cycles of: heat denaturation at95° C. for 20 seconds, annealing at 60° C. for 20 seconds, elongation at72° C. for 20 seconds, as well as heat denaturation at 95° C. for 5minutes prior to repeating the reaction and finally elongation at 72° C.for 10 minutes. After completing the first PCR reaction, nested PCR wascarried out. One μl of the product of the first PCR was used as atemplate, whilst primers employed were 2RC2 Primer(5′-gtacgccacagcgtatgatgc-3′ (SEQ ID NO: 19)) as attached, and syntheticTGP2 Primer (5′-cattcttgacaaacttcatag-3′ (SEQ ID NO: 20)), which wassynthesized similarly to TGP1 Primer. The reaction was conducted with asimilar constitution of the reaction and program to those of the firstPCR, except that cycle number was 25 cycles. Such a PCR reaction wascarried out with TaKaRa PCR Thermal Cycler 480 manufactured by TakaraShuzo Co., Ltd. Thus resulting PCR product was confirmed on agarose gelelectrophoresis. Thereafter, the band was excised from the gel, followedby freezing at −80° C. for 10 minutes and centrifugation at 15,000 rpmfor 10 minutes. After the centrifugation, the supernatant wasprecipitated with ethanol. Accordingly, the purification wasaccomplished.

[0188] Purified DNA fragment was incorporated into pT7Blue Vectormanufactured by Novagen Co., and thus resulting vector was transformedinto competent XL1-Blue cells. The transformant was cultured in LBmedium (100 μg/ml ampicillin), and then a plasmid was extracted by analkali SDS method. Nucleotide sequence was determined with AutoReadSequencing Kit and A. L. F. DNA Sequencer manufactured by Pharmacia Co.Primers employed were M13 Universal Primer (SEQ ID NO: 7) and M13Reverse Primer (SEQ ID NO: 8) attached to AutoRead Sequencing Kit.

[0189] Additionally, screening of a cDNA library derived from placenta(Clontech Co.,) was carried out after synthesizing a primer:5′-atcttgctgcagattcgtgac-3′ (SEQ ID NO: 21), corresponding to theupstream direction from the sequence of an N terminal portion of thecDNA clone obtained for the confirmation of the N terminus. Thescreening was carried out by PCR using a primer synthesizedcorresponding to the upstream direction: 5′-atcttgctgcagattcgtgac-3′(SEQ ID NO: 21) and a primer λgt11 5′ Sequencing Primer:5′-gactcctggagcccg-3′ (SEQ ID NO: 22) that is a part included in thevector. The reaction mixture was prepared by adding 2.5 mM MgCl₂, 1×LAPCR Buffer II (Mg²⁺ free), 2U TaKaRa LA Taq, two kinds of primers (each0.2 μM 5′-atcttgctgcagattcgtgac-3′ (SEQ ID NO: 21), λgt11 5′ SequencingPrimer: 5′-gactcctggagcccg-3′ (SEQ ID NO: 22)), 1 μl of a cDNA libraryderived from placenta to water to give the total volume of 50 μl. Thereaction was executed with one cycle of at 94° C. for 2 minutes, and 50cycles of at 94° C. for 30 seconds, at 60° C. for 30 seconds, and at 72°C. for 1 minute and 30 seconds.

[0190] Thus resulting cDNA was separated on agarose gel electrophoresis,and stained with a solution of ethidium bromide (0,1 μg/ml). Uponconfirmation of the migration pattern with a trans illuminator, theamplification of an insert corresponding to about 600 bp wasdemonstrated. Then, the amplified part was excised from the agarose gel,followed by freezing at −80° C. for 10 minutes and centrifugation at15,000 rpm for 10 minutes. After the centrifugation, the supernatant wasrecovered and subjected to the precipitation with ethanol. Accordingly,the purification was accomplished. Purified DNA fragment wasincorporated into pT7Blue Vector manufactured by Novagen Co., and thusresulting vector was transformed into competent XL1-Blue cells. Thetransformant was cultured in LB medium (50 μg/ml ampicillin), and then aplasmid was extracted by an alkali SDS method. Nucleotide sequence wasdetermined with DNA Sequencing Kit and Sequencer ABI PRISM 377manufactured by PE Applied Biosystems Inc. Primers employed were M13Universal Primer (SEQ ID NO: 7) and M13 Reverse Primer (SEQ ID NO: 8)attached to AutoRead Sequencing kit manufactured by Pharmacia Co.

[0191] Consequently, it was revealed that the sequence contained further604 bases long from the nucleotide sequence that had been obtained tothe N-terminal direction. Accordingly, it was confirmed that theresultant cDNA of hSRCL-P1 obtained hereby includes 2628 bases, havingORF (open reading frame) of 2226 bases (SEQ ID NO: 1), which encodesamino acid sequence of 742 amino acids set out in SEQ ID NO: 2.

[0192] Next, search on the homology for DNA and amino acid on GenBankdatabase was conducted. As a result, the resulting amino acid sequencewas revealed to be that of a novel protein, which is distinct from anycollectin/scavenger receptors that have been found so far.

[0193] Furthermore, a mutant was obtained, having deletion of aminoacids position 483 to 606 of the amino acid sequence set out in SEQ IDNO: 2, encoded by the nucleotide sequences position 74 to 1993 set outin SEQ ID NO: 23.

Example 5 Obtaining cDNA of Mouse Novel Scavenger Receptor

[0194] In a similar manner to that for hSRCL-P1, mSRCL-P1 gene could beobtained through the screening of a mouse liver cDNA library. Theresulting cDNA clone of mSRCL-P1 includes 2637 bases, having ORF (openreading frame) of 2226 bases (SEQ ID NO: 3), which was confirmed toencode amino acid sequence of 742 amino acids set out in SEQ ID NO: 4.

Example 6 Construction of Transient Expression Vector of hSRCL-P1,pEGFP-N1-hSRCL-P1

[0195] Amplification of hSRCL-P1 from initiation codon to terminationcodon, set out in SEQ ID NO: 1 was carried out first using a primerconsisting of the nucleotide sequence: ccgctcgagcggtcaccatgaaagacgact(SEQ ID NO: 25) and a primer consisting of the nucleotide sequence:tccccgcggtaatgcagatgacagtactgt (SEQ ID NO: 26) with a cDNA libraryderived from human placenta as a template by PCR (manufactured by TakaraKK: Takara Thermal Cycler MP). Thus resulting hSRCL-P1cDNA was ligatedto pT7Blue T-Vector (Novagen Co.), and transformed into Escherichiacoli, XL1-Blue. A plasmid containing hSRCL-P1cDNA was purified from theresulting clone. Following the confirmation of the nucleotide sequenceof the resulting plasmid with a sequencer, the plasmid with no error wasdigested with restriction enzymes Xho I and Sac II, and ligated topEGFP-N1 vector (Clontech Co.) that had been digested with the sameenzymes and purified. After the ligated plasmid was transformed intoEscherichia coli, XL1-Blue, the resulting clone was cultured. Theplasmid was then purified to give a transient expression vectorpEGFP-N1-hSRCL-P1.

Example 7 Expression of hSRCL-P1 Using a Transient Expression System

[0196] Transient expression in CHO cells was attempted using theexpression vector pEGFP-N1-hSRCL-P1 obtained in Example 6, andLIPOFECTAMINE 2000 (LF2000) Reagent (GIBCO BRL Co.). A solution of 0.2ml of LF2000 Reagent (LF2000 Reagent 12 μl, Nutrient Mixture F-12 Ham(Ham's F-12 medium, (manufactured by Sigma Co.))) was first prepared,and incubated at room temperature for 5 minutes. Then, 0.2 ml of avector solution (pEGFP-N1-hSRCL-P1 vector 4 μg, Ham's F-12 medium) wasadmixed therewith, followed by the incubation for 20 minutes.Thereafter, the solution was added to CHO cells that had been culturedto a high density in a 35 mm dish containing 2 ml of Ham's F-12 medium(containing 5% FCS). After incubating at 37° C. for 4 hours in thepresence of 5% CO₂, the medium was replaced with a flesh medium,followed by subsequent incubation at 37° C. for 20 hours in the presenceof 5% CO₂. The presence or absence of the expression could be confirmedby the observation of the fluorescent image for GFP by a fluorescenceobservation system of an inverted system microscope IX70 manufactured byOlympus Co., Ltd. Thus resultant cells were identified as cellstransiently expressing hSRCL-P1.

Example 8 Construction of a Vector pcDNA3.1/Myc-His A-hSRCL-P1 forProducing Cell Strain Stably Expressing hSRCL-P1

[0197] Amplification of hSRCL-P1, from initiation codon to terminationcodon, set out in SEQ ID NO: 1 was carried out first using a primerconsisting of the nucleotide sequence:aatgcggccgcaccatgaaagacgacttcgcagag (SEQ ID NO: 27) and a primerconsisting of the nucleotide sequence: gctctagaccgcggtaatgcagatgacagtac(SEQ ID NO: 28) with a cDNA library derived from human placenta as atemplate by PCR (manufactured by Takara KK: Takara Thermal Cycler MP).Thus resulting hSRCL-P1cDNA was ligated to pT7Blue T-Vector (NovagenCo.), and transformed into Escherichia coli, XLI-Blue. A plasmidcontaining hSRCL-P1cDNA was purified from the resulting clone. Followingthe confirmation of the nucleotide sequence of the resulting plasmidwith a sequencer, the plasmid with no error was digested withrestriction enzymes Not I and Sac II, and ligated to pcDNA3.1/Myc-His Avector (Invitorogen Co.) that had been digested with the same enzymesand purified. After the ligated plasmid was transformed into Escherichiacoli, XL1-Blue, the resulting clone was cultured. The plasmid was thenpurified to give vector pcDNA3.1/Myc-His A-hSRCL-P1 for producing astable cell strain.

Example 9 Preparation of Cell Strain Stably Expressing hSRCL-P1

[0198] Stable expression of hSRCL-P1 was attempted using the expressionvector pcDNA3.1/Myc-His A-hSRCL-P1 obtained in Example 8, andLIPOFECTAMINE 2000 (LF2000) Reagent (GIBCO BRL Co.). A 0.5 ml solutionof LF2000 Reagent (LF2000 Reagent 30 μl, Ham's F-12 medium) was firstprepared, and incubated at room temperature for 5 minutes. Then, 0.5 mlof a vector solution (vector 10 μg, Nutrient Mixture F-12 Ham (Ham'sF-12 medium) (Sigma Co.)) was admixed therewith, followed by theincubation for 20 minutes. Thereafter, the solution was added to CHOcells that had been cultured to a high density in a 25 cm² flaskcontaining 5 ml of Ham's F-12 medium (containing 5% FCS). Afterincubating at 37° C. for 4 hours in the presence of 5% CO₂, the mediumwas replaced with a flesh medium, followed by subsequent incubation at37° C. for 20 hours in the presence of 5% CO₂. Next, the medium wasreplaced with Ham's F-12 medium containing 5% FCS, 0.4 mg/ml Geneticin(GIBCO BRL Co.), and 10 days culture was subsequently conducted. In themethod, the medium was replaced once.

[0199] Through this selection by a drug for 10 days, only thetransformed cells could survive and proliferated, however to thecontrary, cells that were not transformed died. In order to obtainhighly expressing cells from the resulting transformed cells, sortingwas performed by a cell sorter (Becton Dickinson Co.). Staining ofhSRCL-P1 expressed on the cell surface was first conducted. Afterwashing the transformed cells in the 25 cm² flask with 5 ml PBS(−)twice, the cells were unstuck with 0.3 ml of 0.02% EDTA solution(Nakarai Tesc KK). The cells were suspended in 10 ml PBS (−), andthereafter centrifuged at 200×g for 7 minutes at 4° C. to remove thesupernatant. To the remaining cells, added 50 μl of a solution of ananti-myc antibody (Invitorogen Co.) that was diluted with 2% FCS/PBS (−)by ten folds. After intimately suspending the cells, the suspension wasincubated at 4° C. for 20 minutes. Thereafter, 10 ml of 2% FCS/PBS (−)was added thereto and suspended, followed by washes through thecentrifugation at 200×g for 7 minutes at 4° C. and the removal of thesupernatant. To the remaining cells, added 50 μl of a solution of asecondary antibody Alexa488 labeled anti-mouse IgG (H+L) that wasdiluted with 2% FCS/PBS (−) by ten folds. After intimately suspendingthe cells, the suspension was incubated at 4° C. for 20 minutes.Thereafter, 10 ml of 2% FCS/PBS (−) was added thereto and suspended,followed by washes through the centrifugation at 200×g for 7 minutes at4° C. and the removal of the supernatant. The remaining cells weresuspended in 0.5 ml of 2% FCS/PBS (−) to give a sorting sample. Afterthe sample was passed through a 5 ml tube equipped with a cell strainercap (Becton Dickinson Co.), it was applied to a cell sorter. CHO cellswithout subjecting to the transformation, which had been similarlytreated, were used as control cells. Accordingly, a sample was selected,which exhibits fluorescence intensity of 10 times or greater than thecontrol sample.

[0200] These cells were dispensed into 96-well cell culture plates, ofwhich wells respectively contained 100 μl Ham's F-12 medium (containing5% FCS, 0.4 mg/ml Geneticin), to charge a single cell per well. Afterthe cells were cultured at 37° C. in the presence of 5% CO₂ for oneweek, additionally each 100 μl of a culture medium was added theretofollowed by the additional culture for one week. A clone proliferated inthe drug selection with Geneticin was divided into two parts, which weresubjected to passages on 12-well and 24-well cell culture plates. Uponthe passage, clones that proliferated from two cells or more per wellwere excluded, and the cells were plated at a cell number ratio of 9:1to 12-well and 24-well cell culture plates. The cells were cultured at37° C. in the presence of 5% CO₂ until the cells in the 12-well platereach to high density. Then, the cells were stained again similarly tothe procedure where individual clones were subjected to sorting, andthereafter, they were applied to FACSCalibur (Becton Dickinson Co.) todetermine the expression level. After determining a clone exhibiting theexpression at a higher amount, respectively corresponding cells in the24-well plate clone were identified as a stable expression cell strain(CHO/hSRCL-P1).

Example 10 Binding Specificity of hSRCL-P1

[0201] Binding specificity of hSRCL-P1 was examined using the stableexpression cell strain CHO/hSRCL-P1, which was obtained in Example 9,for (1) yeast (Zymosan A Bioparticles, manufactured by Molecular ProbesCo.); gram negative bacterium (Escherichia coli Bioparticles,manufactured by Molecular Probes Co.); or gram positive bacterium(Staphylococcus aureus Bioparticles, manufactured by Molecular ProbesCo.), (2) oxidized LDL (2.0 mg/ml LDL added with 50 μM CuSO₄ followed bysubjecting to the reaction for 24 hours and to the dialysis in PBS(−)),(3) AGE-HSA (AGE-human serum albumin, which was prepared according toIkeda, K. et al., Biochemistry 35(24), 8075-8083 (1996)), or (4) mannose(α-D-Mannose BP-Probe, manufactured by Seikagaku Kogyo KK) or fucose(α-L-Fucose BP-Probe, manufactured by Seikagaku Kogyo KK).

[0202] CHO/hSRCL-P1 was first plated on a 35 mm bottom dish (Matsunamiglass KK) at 1×10⁵ cells, and cultured at 37° C. for 3 days in thepresence of 5% CO₂. The culture was conducted in 2 ml of Ham's F-12medium containing 5% FCS, 0.4 mg/ml Geneticin. After 3 days passed, thecells were washed twice with 1 ml of Minimum Essential Medium AlphaMedium containing 2% FCS (αMEM/2% FCS). Thereafter, each 1 ml of αMEM/2%FCS containing 25 μg/ml yeast, 25 μg/ml gram negative bacterium, 25μg/ml gram positive bacterium, 5 μg/ml oxidized LDL, 10 μg/ml AGE, or 10μg/ml mannose or 10 μg/ml fucose was added thereto. Three hours reactionat 4° C. was allowed, and thereafter, the cells were washed five timeswith 1 ml of αMEM/2% FCS.

[0203] The binding was confirmed as below. First, in regard to (2), (3)and (4), each 1 ml of 100 folds dilution of (2) an anti-oxidizedphosphatidylcholine antibody; (3) an anti-HSA antibody (BIOSYS Co.); or(4) streptavidin, Alexa594 conjugate (Molecular Probes Co.) in αMEM/2%FCS was added, followed by further 30 minutes incubation at 4° C.Thereafter, the cells were washed three times with 1 ml of αMEM/2% FCS.Next, 0.2 ml of a solution of 4% paraformaldehyde/PBS (−) was added toany one of the above (1) to (4), and then the fixation was allowed bythe incubation at room temperature for 20 minutes. Then 1 ml of TBSC(Takara Shuzo Co., Ltd, a buffer containing TBS (Tris-Buffered Saline)Powder which was adjusted to give a predetermined amount with sterilizedand distilled water, and added with CaCl₂ at a final concentration of 5mM) was used for three times washes. Next, in regard to (2) and (3), thereaction with secondary antibody was performed. More specifically, each1 ml of 200 folds dilution of (2) rhodamine labeled anti-mouse IgM MuChain (Chemicon International Co.); or (3) Alexa 594 anti-goat IgG (H+L)(Molecular Probes Co.) in 25% BlockAce (Dainippon Pharmaceutical Co.,Ltd.)/TBSC was added thereto, followed by further 30 minutes incubationat room temperature. Thereafter, the cells were washed three times with1 ml of TBSC. Next, in regard to from (1) to (4), SlowFade LightAntifade Kit (Molecular Probes Co.) was used for the mounting to givesamples for the observation under a fluorescence microscope. For each ofthe samples, the fluorescent image was observed by a fluorescenceobservation system of an inverted system microscope IX70 manufactured byOlympus Co., Ltd. The results are respectively depicted in FIG. 5 for(1) (A: yeast, B: gram negative bacterium (Escherichia coli), and C:gram positive bacterium (Staphylococcus aureus)), and in FIG. 6 for(2)-(4) (A: oxidized LDL, B: mannose, and C: AGE). As is clear fromthese figures, specific binding images could be observed in CHO cellsthat are stably expressing hSRCL-P1 in all cases of from (1) to (4). Theresults shown in FIG. 5, A-C, in which bacterium was employed, clearlyindicated that each of the stained parts of hSRCL-P1 (in each leftfigure, stained in green) overlapped with each of the parts where thebacteria are present (in each middle figure, stained in red), which wasfound as Overlap in each right figure, and that each of the bacteriaspecifically bound to hSRCL-P1.

[0204] In addition, cells in which hSRCL-P1 was transiently expressed(see, Example 7) gave the similar results, which demonstrate thespecific binding.

Example 11 Intracellular Incorporation of Binding Complex byPhagocytosis of hSRCL-P1

[0205] Intracellular incorporation of each binding complex, which wasemployed in Example 10, was observed using the transient expression celland stable expression cell strains of hSRCL-P1 obtained in Examples 7and 9. Incorporation of the binding complex was confirmed by modifyingmethod described in Example 10, in which the reaction with the bindingcomplex was performed at a temperature of 37° C. After staining, theincorporation status within the cells was observed by three-dimensionalimage processing using a confocal laser scanning microscope manufacturedby Olympus Co., Ltd. The results in the examination where the transientexpression cells were used are depicted in FIG. 7 in regard to thoseobtained for yeast, which reveal that yeast cells (stained in red) wereincorporated into cells that were expressing hSRCL-P1 (stained ingreen). Furthermore, similar results were also obtained when the stableexpression cell strain was employed.

Example 12 Demonstration of SRCL-P1 Expression in Vascular EndothelialCells

[0206] In order to verify the expression and localization of hSRCL-P1 intissues, fluorescent immunostaining was performed in accordance with themanipulation below using paraffin embedded sections derived from healthyhuman and mouse heart (Novagen Co.).

[0207] Slides with the paraffin embedded section was immersed 3 times inxylene at room temperature for 10 minutes in a stain tray to effect theparaffin removal treatment. Thereafter, the slides were sequentiallyimmersed in 100%-90%-80%-70% -ethanol at room temperature for 10 minuteseach, and into PBS (−) solution for 10 minutes for achieving thehydration treatment.

[0208] Next, the slides were immersed in a solution of PBS (−)containing 3% hydrogen peroxide in order to suppress the peroxidaseactivity that intrinsically exists on the tissue section at roomtemperature for 10 minutes. Thereafter, blocking was carried out byimmersing the slide in Blocking Ace (Dainippon Pharmaceutical Co., Ltd.)at room temperature for 1 hour.

[0209] Next, 100 μl of an anti-hSRCL-P1 rabbit polyclonal antibody (IgGfraction, 100 μg/ml) as a primary antibody was applied on the tissuesection in a humid box, and subjected to a reaction for 30 minutes. Theprimary antibody was washed three times by immersing into a washingsolution (Tris-HCl: pH 7.5, 0.15 M NaCl, 0.05% Tween 20) in a stainingtray while shaking for 10 minutes at room temperature. Thereafter, POD(peroxidase) labeled anti-rabbit IgG sheep antibody (Boeringer MannheimCo., Ltd.) as a secondary antibody was reacted in a similar manner tothe primary antibody at a concentration of 5 U/ml, followed by washes.Then, Biotinyl Tyramide Amplification Reagent (NEN (trade name),manufactured by Life Science Products Co.) was applied to the slide, andthe reaction was allowed at room temperature for 10 minutes, followed bysimilar washes to the procedure for the primary antibody. Avidin AlexaFluor (trade name) 488 conjugate (manufactured by Molecular Probes Co.)of 1 mg/ml was diluted to 100-folds in a solution of PBS (−), and 100 μlof the resulting solution was applied to the tissue section on the slidein the humid box at room temperature, and subjected to a reaction for 30minutes, followed by similar washes to the procedure for the primaryantibody. Thereafter, Slow Fade Light Antifade Kit (Molecular ProbesCO.) was used for the mounting to give a sample for the observation witha fluorescence microscope (Nikon Co.). In addition, a slide for thenegative control was prepared in a similar manner except that normalrabbit serum was used for the reaction instead of the primary antibody.

[0210] Consequently, stained images were observed in heart vascularendothelial cells for both of A: healthy human and B: mouse as shown inFIG. 8 (left figure each), while such stained images were not foundwhatever in the negative control (right figure each). Accordingly, itwas verified that SRCL-P1 was expressed in vascular endothelial cells inthe heart, suggesting that SRCL-P1 participates in the binding ofoxidized LDL, AGE and the like onto the blood vessel wall.

EFFECT OF THE INVENTION

[0211] Because SRCL-P1 protein of the present invention has an SRstructure and a collectin structure, it is believed to be a substancethat exerts characteristic effects to those structures. Therefore, itcan be utilized in the elucidation of mechanisms of macrophage and basicimmunity; in the elucidation of mechanisms of the development of a widevariety of diseases such as arteriosclerosis, diabetic complications andAlzheimer's disease, hyper β-lipoproteinemia, hypercholesterolemia,hypertriglyceridemia, hypo α-lipoproteinemia, transplantation,atherectomy, post angiogenic restenosis, bacterial infections; in thediagnostic, prophylactic and therapeutic methods thereof; and in thedevelopment of reagents and drugs for the same.

1 28 1 2628 DNA Homo Sapiens CDS (74)..(2299) 1 ggggggacga cttcctcggctgcgcggcgc tcgcgcggag ctccccggcc ggcggtgcgt 60 ccccacggtc acc atg aaagac gac ttc gca gag gag gag gag gtg caa 109 Met Lys Asp Asp Phe Ala GluGlu Glu Glu Val Gln 1 5 10 tcc ttc ggt tac aag cgg ttt ggt att cag gaagga aca caa tgt acc 157 Ser Phe Gly Tyr Lys Arg Phe Gly Ile Gln Glu GlyThr Gln Cys Thr 15 20 25 aaa tgt aaa aat aac tgg gca ctg aag ttt tct atcata tta tta tac 205 Lys Cys Lys Asn Asn Trp Ala Leu Lys Phe Ser Ile IleLeu Leu Tyr 30 35 40 att ttg tgt gcc ttg cta aca atc aca gta gcc att ttggga tat aaa 253 Ile Leu Cys Ala Leu Leu Thr Ile Thr Val Ala Ile Leu GlyTyr Lys 45 50 55 60 gtt gta gag aaa atg gac aat gtc aca ggt ggc atg gaaaca tct cgc 301 Val Val Glu Lys Met Asp Asn Val Thr Gly Gly Met Glu ThrSer Arg 65 70 75 caa acc tat gat gac aag ctc aca gca gtg gaa agt gac ctgaaa aaa 349 Gln Thr Tyr Asp Asp Lys Leu Thr Ala Val Glu Ser Asp Leu LysLys 80 85 90 tta ggt gac caa act ggg aag aaa gct atc agc acc aac tca gaactc 397 Leu Gly Asp Gln Thr Gly Lys Lys Ala Ile Ser Thr Asn Ser Glu Leu95 100 105 tcc acc ttc aga tca gac att cta gat ctc cgt cag caa ctt cgtgag 445 Ser Thr Phe Arg Ser Asp Ile Leu Asp Leu Arg Gln Gln Leu Arg Glu110 115 120 att aca gaa aaa acc agc aag aac aag gat acg ctg gag aag ttacag 493 Ile Thr Glu Lys Thr Ser Lys Asn Lys Asp Thr Leu Glu Lys Leu Gln125 130 135 140 gcg agc ggg gat gct ctg gtg gac agg cag agt caa ttg aaagaa act 541 Ala Ser Gly Asp Ala Leu Val Asp Arg Gln Ser Gln Leu Lys GluThr 145 150 155 ttg gag aat aac tct ttc ctc atc acc act gta aac aaa accctc cag 589 Leu Glu Asn Asn Ser Phe Leu Ile Thr Thr Val Asn Lys Thr LeuGln 160 165 170 gcg tat aat ggc tat gtc acg aat ctg cag caa gat acc agcgtg ctc 637 Ala Tyr Asn Gly Tyr Val Thr Asn Leu Gln Gln Asp Thr Ser ValLeu 175 180 185 cag ggc aat ctg cag aac caa atg tat tct cat aat gtg gtcatc atg 685 Gln Gly Asn Leu Gln Asn Gln Met Tyr Ser His Asn Val Val IleMet 190 195 200 aac ctc aac aac ctg aac ctg acc cag gtg cag cag agg aacctc atc 733 Asn Leu Asn Asn Leu Asn Leu Thr Gln Val Gln Gln Arg Asn LeuIle 205 210 215 220 acg aat ctg cag cgg tct gtg gat gac aca agc cag gctatc cag cga 781 Thr Asn Leu Gln Arg Ser Val Asp Asp Thr Ser Gln Ala IleGln Arg 225 230 235 atc aag aac gac ttt caa aat ctg cag cag gtt ttt cttcaa gcc aag 829 Ile Lys Asn Asp Phe Gln Asn Leu Gln Gln Val Phe Leu GlnAla Lys 240 245 250 aag gac acg gat tgg ctg aag gag aaa gtg cag agc ttgcag acg ctg 877 Lys Asp Thr Asp Trp Leu Lys Glu Lys Val Gln Ser Leu GlnThr Leu 255 260 265 gct gcc aac aac tct gcg ttg gcc aaa gcc aac aac gacacc ctg gag 925 Ala Ala Asn Asn Ser Ala Leu Ala Lys Ala Asn Asn Asp ThrLeu Glu 270 275 280 gat atg aac agc cag ctc aac tca ttc aca ggt cag atggag aac atc 973 Asp Met Asn Ser Gln Leu Asn Ser Phe Thr Gly Gln Met GluAsn Ile 285 290 295 300 acc act atc tct caa gcc aac gag cag aac ctg aaagac ctg cag gac 1021 Thr Thr Ile Ser Gln Ala Asn Glu Gln Asn Leu Lys AspLeu Gln Asp 305 310 315 tta cac aaa gat gca gag aat aga aca gcc atc aagttc aac caa ctg 1069 Leu His Lys Asp Ala Glu Asn Arg Thr Ala Ile Lys PheAsn Gln Leu 320 325 330 gag gaa cgc ttc cag ctc ttt gag acg gat att gtgaac atc att agc 1117 Glu Glu Arg Phe Gln Leu Phe Glu Thr Asp Ile Val AsnIle Ile Ser 335 340 345 aat atc agt tac aca gcc cac cac ctg cgg acg ctgacc agc aat cta 1165 Asn Ile Ser Tyr Thr Ala His His Leu Arg Thr Leu ThrSer Asn Leu 350 355 360 aat gaa gtc agg acc act tgc aca gat acc ctt accaaa cac aca gat 1213 Asn Glu Val Arg Thr Thr Cys Thr Asp Thr Leu Thr LysHis Thr Asp 365 370 375 380 gat ctg acc tcc ttg aat aat acc ctg gcc aacatc cgt ttg gat tct 1261 Asp Leu Thr Ser Leu Asn Asn Thr Leu Ala Asn IleArg Leu Asp Ser 385 390 395 gtt tct ctc agg atg caa caa gat ttg atg aggtcg agg tta gac act 1309 Val Ser Leu Arg Met Gln Gln Asp Leu Met Arg SerArg Leu Asp Thr 400 405 410 gaa gta gcc aac tta tca gtg att atg gaa gaaatg aag cta gta gac 1357 Glu Val Ala Asn Leu Ser Val Ile Met Glu Glu MetLys Leu Val Asp 415 420 425 tcc aag cat ggt cag ctc atc aag aat ttt acaata cta caa ggt cca 1405 Ser Lys His Gly Gln Leu Ile Lys Asn Phe Thr IleLeu Gln Gly Pro 430 435 440 ccg ggc ccc agg ggt cca aga ggt gac aga ggatcc cag gga ccc cct 1453 Pro Gly Pro Arg Gly Pro Arg Gly Asp Arg Gly SerGln Gly Pro Pro 445 450 455 460 ggc cca act ggc aac aag gga cag aaa ggagag aag ggg gag cct gga 1501 Gly Pro Thr Gly Asn Lys Gly Gln Lys Gly GluLys Gly Glu Pro Gly 465 470 475 cca cct ggc cct gcg ggt gag aga ggc ccaatt gga cca gct ggt ccc 1549 Pro Pro Gly Pro Ala Gly Glu Arg Gly Pro IleGly Pro Ala Gly Pro 480 485 490 ccc gga gag cgt ggc ggc aaa gga tct aaaggc tcc cag ggc ccc aaa 1597 Pro Gly Glu Arg Gly Gly Lys Gly Ser Lys GlySer Gln Gly Pro Lys 495 500 505 ggc tcc cgt ggt tcc cct ggg aag ccc ggccct cag ggc ccc agt ggg 1645 Gly Ser Arg Gly Ser Pro Gly Lys Pro Gly ProGln Gly Pro Ser Gly 510 515 520 gac cca ggc ccc ccg ggc cca cca ggc aaagag gga ctc ccc ggc cct 1693 Asp Pro Gly Pro Pro Gly Pro Pro Gly Lys GluGly Leu Pro Gly Pro 525 530 535 540 cag ggc cct cct ggc ttc cag gga cttcag ggc acc gtt ggg gag cct 1741 Gln Gly Pro Pro Gly Phe Gln Gly Leu GlnGly Thr Val Gly Glu Pro 545 550 555 ggg gtg cct gga cct cgg gga ctg ccaggc ttg cct ggg gta cca ggc 1789 Gly Val Pro Gly Pro Arg Gly Leu Pro GlyLeu Pro Gly Val Pro Gly 560 565 570 atg cca ggc ccc aag ggc ccc ccc ggccct cct ggc cca tca gga gcg 1837 Met Pro Gly Pro Lys Gly Pro Pro Gly ProPro Gly Pro Ser Gly Ala 575 580 585 gtg gtg ccc ctg gcc ctg cag aat gagcca acc ccg gca ccg gag gac 1885 Val Val Pro Leu Ala Leu Gln Asn Glu ProThr Pro Ala Pro Glu Asp 590 595 600 aat ggc tgc ccg cct cac tgg aag aacttc aca gac aaa tgc tac tat 1933 Asn Gly Cys Pro Pro His Trp Lys Asn PheThr Asp Lys Cys Tyr Tyr 605 610 615 620 ttt tca gtt gag aaa gaa att tttgag gat gca aag ctt ttc tgt gaa 1981 Phe Ser Val Glu Lys Glu Ile Phe GluAsp Ala Lys Leu Phe Cys Glu 625 630 635 gac aag tct tca cat ctt gtt ttcata aac act aga gag gaa cag caa 2029 Asp Lys Ser Ser His Leu Val Phe IleAsn Thr Arg Glu Glu Gln Gln 640 645 650 tgg ata aaa aaa cag atg gta gggaga gag agc cac tgg atc ggc ctc 2077 Trp Ile Lys Lys Gln Met Val Gly ArgGlu Ser His Trp Ile Gly Leu 655 660 665 aca gac tca gag cgt gaa aat gaatgg aag tgg ctg gat ggg aca tct 2125 Thr Asp Ser Glu Arg Glu Asn Glu TrpLys Trp Leu Asp Gly Thr Ser 670 675 680 cca gac tac aaa aat tgg aaa gctgga cag ccg gat aac tgg ggt cat 2173 Pro Asp Tyr Lys Asn Trp Lys Ala GlyGln Pro Asp Asn Trp Gly His 685 690 695 700 ggc cat ggg cca gga gaa gactgt gct ggg ttg att tat gct ggg cag 2221 Gly His Gly Pro Gly Glu Asp CysAla Gly Leu Ile Tyr Ala Gly Gln 705 710 715 tgg aac gat ttc caa tgt gaagac gtc aat aac ttc att tgc gaa aaa 2269 Trp Asn Asp Phe Gln Cys Glu AspVal Asn Asn Phe Ile Cys Glu Lys 720 725 730 gac agg gag aca gta ctg tcatct gca tta taacggactg tgatgggatc 2319 Asp Arg Glu Thr Val Leu Ser SerAla Leu 735 740 acatgagcaa attttcagct ctcaaaggca aaggacactc ctttctaattgcatcacctt 2379 ctcatcagat tgaaaaaaaa aaaagcactg aaaaccaatt actgaaaaaaaattgacagc 2439 tagtgttttt taccatccgt cattacccaa agacttggga actaaaatgttccccagggt 2499 gatatgctga ttttcattgt gcacatggac tgaatcacat agattctcctccgtcagtaa 2559 ccgtgcgatt atacaaatta tgtcttccaa agtatggaac actccaatcagaaaaaggtt 2619 atcatcccg 2628 2 742 PRT Homo Sapiens Deduced Amino AcidSequence of Novel Human Scavenger Receptor from Nucleotide Sequence. 2Met Lys Asp Asp Phe Ala Glu Glu Glu Glu Val Gln Ser Phe Gly Tyr 1 5 1015 Lys Arg Phe Gly Ile Gln Glu Gly Thr Gln Cys Thr Lys Cys Lys Asn 20 2530 Asn Trp Ala Leu Lys Phe Ser Ile Ile Leu Leu Tyr Ile Leu Cys Ala 35 4045 Leu Leu Thr Ile Thr Val Ala Ile Leu Gly Tyr Lys Val Val Glu Lys 50 5560 Met Asp Asn Val Thr Gly Gly Met Glu Thr Ser Arg Gln Thr Tyr Asp 65 7075 80 Asp Lys Leu Thr Ala Val Glu Ser Asp Leu Lys Lys Leu Gly Asp Gln 8590 95 Thr Gly Lys Lys Ala Ile Ser Thr Asn Ser Glu Leu Ser Thr Phe Arg100 105 110 Ser Asp Ile Leu Asp Leu Arg Gln Gln Leu Arg Glu Ile Thr GluLys 115 120 125 Thr Ser Lys Asn Lys Asp Thr Leu Glu Lys Leu Gln Ala SerGly Asp 130 135 140 Ala Leu Val Asp Arg Gln Ser Gln Leu Lys Glu Thr LeuGlu Asn Asn 145 150 155 160 Ser Phe Leu Ile Thr Thr Val Asn Lys Thr LeuGln Ala Tyr Asn Gly 165 170 175 Tyr Val Thr Asn Leu Gln Gln Asp Thr SerVal Leu Gln Gly Asn Leu 180 185 190 Gln Asn Gln Met Tyr Ser His Asn ValVal Ile Met Asn Leu Asn Asn 195 200 205 Leu Asn Leu Thr Gln Val Gln GlnArg Asn Leu Ile Thr Asn Leu Gln 210 215 220 Arg Ser Val Asp Asp Thr SerGln Ala Ile Gln Arg Ile Lys Asn Asp 225 230 235 240 Phe Gln Asn Leu GlnGln Val Phe Leu Gln Ala Lys Lys Asp Thr Asp 245 250 255 Trp Leu Lys GluLys Val Gln Ser Leu Gln Thr Leu Ala Ala Asn Asn 260 265 270 Ser Ala LeuAla Lys Ala Asn Asn Asp Thr Leu Glu Asp Met Asn Ser 275 280 285 Gln LeuAsn Ser Phe Thr Gly Gln Met Glu Asn Ile Thr Thr Ile Ser 290 295 300 GlnAla Asn Glu Gln Asn Leu Lys Asp Leu Gln Asp Leu His Lys Asp 305 310 315320 Ala Glu Asn Arg Thr Ala Ile Lys Phe Asn Gln Leu Glu Glu Arg Phe 325330 335 Gln Leu Phe Glu Thr Asp Ile Val Asn Ile Ile Ser Asn Ile Ser Tyr340 345 350 Thr Ala His His Leu Arg Thr Leu Thr Ser Asn Leu Asn Glu ValArg 355 360 365 Thr Thr Cys Thr Asp Thr Leu Thr Lys His Thr Asp Asp LeuThr Ser 370 375 380 Leu Asn Asn Thr Leu Ala Asn Ile Arg Leu Asp Ser ValSer Leu Arg 385 390 395 400 Met Gln Gln Asp Leu Met Arg Ser Arg Leu AspThr Glu Val Ala Asn 405 410 415 Leu Ser Val Ile Met Glu Glu Met Lys LeuVal Asp Ser Lys His Gly 420 425 430 Gln Leu Ile Lys Asn Phe Thr Ile LeuGln Gly Pro Pro Gly Pro Arg 435 440 445 Gly Pro Arg Gly Asp Arg Gly SerGln Gly Pro Pro Gly Pro Thr Gly 450 455 460 Asn Lys Gly Gln Lys Gly GluLys Gly Glu Pro Gly Pro Pro Gly Pro 465 470 475 480 Ala Gly Glu Arg GlyPro Ile Gly Pro Ala Gly Pro Pro Gly Glu Arg 485 490 495 Gly Gly Lys GlySer Lys Gly Ser Gln Gly Pro Lys Gly Ser Arg Gly 500 505 510 Ser Pro GlyLys Pro Gly Pro Gln Gly Pro Ser Gly Asp Pro Gly Pro 515 520 525 Pro GlyPro Pro Gly Lys Glu Gly Leu Pro Gly Pro Gln Gly Pro Pro 530 535 540 GlyPhe Gln Gly Leu Gln Gly Thr Val Gly Glu Pro Gly Val Pro Gly 545 550 555560 Pro Arg Gly Leu Pro Gly Leu Pro Gly Val Pro Gly Met Pro Gly Pro 565570 575 Lys Gly Pro Pro Gly Pro Pro Gly Pro Ser Gly Ala Val Val Pro Leu580 585 590 Ala Leu Gln Asn Glu Pro Thr Pro Ala Pro Glu Asp Asn Gly CysPro 595 600 605 Pro His Trp Lys Asn Phe Thr Asp Lys Cys Tyr Tyr Phe SerVal Glu 610 615 620 Lys Glu Ile Phe Glu Asp Ala Lys Leu Phe Cys Glu AspLys Ser Ser 625 630 635 640 His Leu Val Phe Ile Asn Thr Arg Glu Glu GlnGln Trp Ile Lys Lys 645 650 655 Gln Met Val Gly Arg Glu Ser His Trp IleGly Leu Thr Asp Ser Glu 660 665 670 Arg Glu Asn Glu Trp Lys Trp Leu AspGly Thr Ser Pro Asp Tyr Lys 675 680 685 Asn Trp Lys Ala Gly Gln Pro AspAsn Trp Gly His Gly His Gly Pro 690 695 700 Gly Glu Asp Cys Ala Gly LeuIle Tyr Ala Gly Gln Trp Asn Asp Phe 705 710 715 720 Gln Cys Glu Asp ValAsn Asn Phe Ile Cys Glu Lys Asp Arg Glu Thr 725 730 735 Val Leu Ser SerAla Leu 740 3 2637 DNA Mouse CDS (92)..(2317) 3 gacgctagga ctggaacgctgaaggctgcc atgggcgtgc agtgagagac actggtacga 60 cttctccggg cggagcgtgtcctcagtcac c atg aaa gac gac ttt gca gag 112 Met Lys Asp Asp Phe Ala Glu1 5 gaa gag gag gtg cag tcc ttc ggt tac aag agg ttt ggt att cag gag 160Glu Glu Glu Val Gln Ser Phe Gly Tyr Lys Arg Phe Gly Ile Gln Glu 10 15 20ggg aca cag tgt acc aaa tgt aaa aat aac tgg gca ctg aag ttt tcg 208 GlyThr Gln Cys Thr Lys Cys Lys Asn Asn Trp Ala Leu Lys Phe Ser 25 30 35 attgta tta tta tac att ctg tgt gcc tta ctg acc atc aca gta gcc 256 Ile ValLeu Leu Tyr Ile Leu Cys Ala Leu Leu Thr Ile Thr Val Ala 40 45 50 55 attttg gga tat aaa gtt gta gag aaa atg gac aat gtc aca gat ggc 304 Ile LeuGly Tyr Lys Val Val Glu Lys Met Asp Asn Val Thr Asp Gly 60 65 70 atg gagaca tct cac cag act tat gac aac aaa ctc act gct gtg gaa 352 Met Glu ThrSer His Gln Thr Tyr Asp Asn Lys Leu Thr Ala Val Glu 75 80 85 agt gac ctgaag aaa tta ggg gat caa gct ggg aag aaa gct cta agt 400 Ser Asp Leu LysLys Leu Gly Asp Gln Ala Gly Lys Lys Ala Leu Ser 90 95 100 acc aac tctgag ctt tct acc ttc aga tca gat att ctg gat ctc cgt 448 Thr Asn Ser GluLeu Ser Thr Phe Arg Ser Asp Ile Leu Asp Leu Arg 105 110 115 caa caa cttcag gag atc aca gaa aaa acc agc aag aac aaa gat acg 496 Gln Gln Leu GlnGlu Ile Thr Glu Lys Thr Ser Lys Asn Lys Asp Thr 120 125 130 135 ctg gagaag ttg caa gca aat ggg gac tca ttg gtt gat agg cag agt 544 Leu Glu LysLeu Gln Ala Asn Gly Asp Ser Leu Val Asp Arg Gln Ser 140 145 150 cag ctgaag gaa act ctg cag aat aat tct ttc ctc att acc acc gtc 592 Gln Leu LysGlu Thr Leu Gln Asn Asn Ser Phe Leu Ile Thr Thr Val 155 160 165 aac aaaaca ctc cag gca tat aat ggc tat gtc aca aat ctg caa caa 640 Asn Lys ThrLeu Gln Ala Tyr Asn Gly Tyr Val Thr Asn Leu Gln Gln 170 175 180 gat actagt gtg ctc cag ggc aat ctg cag agc caa atg tat tct cag 688 Asp Thr SerVal Leu Gln Gly Asn Leu Gln Ser Gln Met Tyr Ser Gln 185 190 195 agc gtggtt atc atg aac ctc aac aac ctg aac cta acc cag gtt cag 736 Ser Val ValIle Met Asn Leu Asn Asn Leu Asn Leu Thr Gln Val Gln 200 205 210 215 cagagg aac ctt atc tca aat ctg cag cag tct gtg gat gac aca agc 784 Gln ArgAsn Leu Ile Ser Asn Leu Gln Gln Ser Val Asp Asp Thr Ser 220 225 230 ctggcc atc cag cga att aag aat gat ttc caa aat ctg cag cag gtt 832 Leu AlaIle Gln Arg Ile Lys Asn Asp Phe Gln Asn Leu Gln Gln Val 235 240 245 ttcctt caa gcc aag aag gac acc gat tgg cta aag gaa aaa gta cag 880 Phe LeuGln Ala Lys Lys Asp Thr Asp Trp Leu Lys Glu Lys Val Gln 250 255 260 agcttg cag aca ttg gct gcc aac aac tct gcc ctg gcc aaa gcc aac 928 Ser LeuGln Thr Leu Ala Ala Asn Asn Ser Ala Leu Ala Lys Ala Asn 265 270 275 aatgac acc cta gag gat atg aat agc cag ctc agc tca ttc aca ggt 976 Asn AspThr Leu Glu Asp Met Asn Ser Gln Leu Ser Ser Phe Thr Gly 280 285 290 295cag atg gac aac att acc act atc tca cag gcc aac gag cag agc ctg 1024 GlnMet Asp Asn Ile Thr Thr Ile Ser Gln Ala Asn Glu Gln Ser Leu 300 305 310aaa gac ctt cag gac tta cac aag gat aca gaa aat aga aca gct gtc 1072 LysAsp Leu Gln Asp Leu His Lys Asp Thr Glu Asn Arg Thr Ala Val 315 320 325aag ttc agc caa ctt gag gaa cgc ttc cag gtc ttt gag aca gat att 1120 LysPhe Ser Gln Leu Glu Glu Arg Phe Gln Val Phe Glu Thr Asp Ile 330 335 340gtg aac atc att agc aac atc agc tac aca gcc cat cac ctg agg aca 1168 ValAsn Ile Ile Ser Asn Ile Ser Tyr Thr Ala His His Leu Arg Thr 345 350 355ctg acc agc aat ctg aat gat gtt agg acc aca tgc aca gac acc ttg 1216 LeuThr Ser Asn Leu Asn Asp Val Arg Thr Thr Cys Thr Asp Thr Leu 360 365 370375 acc aga cac acg gat gac ctg acc tcc ttg aat aac aca cta gtc aac 1264Thr Arg His Thr Asp Asp Leu Thr Ser Leu Asn Asn Thr Leu Val Asn 380 385390 atc cgc ttg gat tct att tct ctc agg atg cag caa gac atg atg agg 1312Ile Arg Leu Asp Ser Ile Ser Leu Arg Met Gln Gln Asp Met Met Arg 395 400405 tca aag tta gac act gaa gtg gcc aac tta tca gtg gtt atg gaa gag 1360Ser Lys Leu Asp Thr Glu Val Ala Asn Leu Ser Val Val Met Glu Glu 410 415420 atg aaa ctg gtt gac tcc aag cac ggt cag ctc atc aag aac ttt acc 1408Met Lys Leu Val Asp Ser Lys His Gly Gln Leu Ile Lys Asn Phe Thr 425 430435 att cta caa ggt cct cct ggc ccc aga ggt cca aaa ggt gac aga gga 1456Ile Leu Gln Gly Pro Pro Gly Pro Arg Gly Pro Lys Gly Asp Arg Gly 440 445450 455 tct cag gga cca cct ggt cca act ggc aac aag gga cag aaa gga gag1504 Ser Gln Gly Pro Pro Gly Pro Thr Gly Asn Lys Gly Gln Lys Gly Glu 460465 470 aag gga gag cct ggt cca cct ggc cct gcg ggt gag agg ggc aca att1552 Lys Gly Glu Pro Gly Pro Pro Gly Pro Ala Gly Glu Arg Gly Thr Ile 475480 485 gga cca gtc ggc cct cct gga gag cgt ggc agc aaa gga tcc aaa ggc1600 Gly Pro Val Gly Pro Pro Gly Glu Arg Gly Ser Lys Gly Ser Lys Gly 490495 500 tca cag ggt ccc aaa gga tct cgt ggg tcc cca ggg aag cct ggc cct1648 Ser Gln Gly Pro Lys Gly Ser Arg Gly Ser Pro Gly Lys Pro Gly Pro 505510 515 caa gga cct agt ggg gac cca gga cca cca ggt cca cca ggc aag gat1696 Gln Gly Pro Ser Gly Asp Pro Gly Pro Pro Gly Pro Pro Gly Lys Asp 520525 530 535 gga ctc cct ggc cct cag ggc cct cct ggc ttc cag gga cta cagggc 1744 Gly Leu Pro Gly Pro Gln Gly Pro Pro Gly Phe Gln Gly Leu Gln Gly540 545 550 act gtg ggt gag cct gga gta cct gga cct cgg ggg ttg cca ggcttg 1792 Thr Val Gly Glu Pro Gly Val Pro Gly Pro Arg Gly Leu Pro Gly Leu555 560 565 cca ggg gtg cca ggc atg cct ggg cct aag gga cca cct ggc cctcca 1840 Pro Gly Val Pro Gly Met Pro Gly Pro Lys Gly Pro Pro Gly Pro Pro570 575 580 ggc ccc tca gga gca atg gag cca ttg gct ctg cag aat gaa ccaacc 1888 Gly Pro Ser Gly Ala Met Glu Pro Leu Ala Leu Gln Asn Glu Pro Thr585 590 595 cca gca tca gag gtc aac gga tgt ccg cct cac tgg aag aac ttcaca 1936 Pro Ala Ser Glu Val Asn Gly Cys Pro Pro His Trp Lys Asn Phe Thr600 605 610 615 gat aaa tgc tac tat ttt tca ttg gaa aaa gaa att ttt gaagat gct 1984 Asp Lys Cys Tyr Tyr Phe Ser Leu Glu Lys Glu Ile Phe Glu AspAla 620 625 630 aag ctt ttc tgt gaa gac aaa tct tcc cat ctc gtt ttc ataaac tca 2032 Lys Leu Phe Cys Glu Asp Lys Ser Ser His Leu Val Phe Ile AsnSer 635 640 645 aga gaa gaa cag caa tgg ata aaa aag cat acc gtg ggg agagaa agc 2080 Arg Glu Glu Gln Gln Trp Ile Lys Lys His Thr Val Gly Arg GluSer 650 655 660 cat tgg atc ggc ctc aca gac tca gaa cag gaa agc gaa tggaag tgg 2128 His Trp Ile Gly Leu Thr Asp Ser Glu Gln Glu Ser Glu Trp LysTrp 665 670 675 cta gac ggg tca cct gtt gat tac aaa aac tgg aaa gct ggacaa cca 2176 Leu Asp Gly Ser Pro Val Asp Tyr Lys Asn Trp Lys Ala Gly GlnPro 680 685 690 695 gat aac tgg ggc agt ggc cat ggg cca gga gaa gac tgtgct ggc ttg 2224 Asp Asn Trp Gly Ser Gly His Gly Pro Gly Glu Asp Cys AlaGly Leu 700 705 710 att tac gca gga cag tgg aat gac ttc cag tgt gat gaaatc aat aac 2272 Ile Tyr Ala Gly Gln Trp Asn Asp Phe Gln Cys Asp Glu IleAsn Asn 715 720 725 ttc att tgt gag aag gaa agg gag gca gta cca tca tccata tta 2317 Phe Ile Cys Glu Lys Glu Arg Glu Ala Val Pro Ser Ser Ile Leu730 735 740 taacagcatg atataatagc agaaacatat tttctgatgc ctctgaaagccgaagaatgc 2377 tcgtttttga ttccatcact tctcaccaga ttgaatggaa aaagctctgaaaagtagtta 2437 ttcaaaataa atggacacct actgcacaat aacccaagga ctagggggctaaaatgctcc 2497 cccaagttga tatattgatt tccagtgtac aaatggactg aatcgcatagattttctcag 2557 ccattaacca tagaatttat gcaaagtata tctttccaaa tatggaatgctccaatcaga 2617 aaaagccaaa aaaaaaaaaa 2637 4 742 PRT Mouse Deduced AminoAcid Sequence of Novel Mouse Scavenger Receptor from NucleotideSequence. 4 Met Lys Asp Asp Phe Ala Glu Glu Glu Glu Val Gln Ser Phe GlyTyr 1 5 10 15 Lys Arg Phe Gly Ile Gln Glu Gly Thr Gln Cys Thr Lys CysLys Asn 20 25 30 Asn Trp Ala Leu Lys Phe Ser Ile Val Leu Leu Tyr Ile LeuCys Ala 35 40 45 Leu Leu Thr Ile Thr Val Ala Ile Leu Gly Tyr Lys Val ValGlu Lys 50 55 60 Met Asp Asn Val Thr Asp Gly Met Glu Thr Ser His Gln ThrTyr Asp 65 70 75 80 Asn Lys Leu Thr Ala Val Glu Ser Asp Leu Lys Lys LeuGly Asp Gln 85 90 95 Ala Gly Lys Lys Ala Leu Ser Thr Asn Ser Glu Leu SerThr Phe Arg 100 105 110 Ser Asp Ile Leu Asp Leu Arg Gln Gln Leu Gln GluIle Thr Glu Lys 115 120 125 Thr Ser Lys Asn Lys Asp Thr Leu Glu Lys LeuGln Ala Asn Gly Asp 130 135 140 Ser Leu Val Asp Arg Gln Ser Gln Leu LysGlu Thr Leu Gln Asn Asn 145 150 155 160 Ser Phe Leu Ile Thr Thr Val AsnLys Thr Leu Gln Ala Tyr Asn Gly 165 170 175 Tyr Val Thr Asn Leu Gln GlnAsp Thr Ser Val Leu Gln Gly Asn Leu 180 185 190 Gln Ser Gln Met Tyr SerGln Ser Val Val Ile Met Asn Leu Asn Asn 195 200 205 Leu Asn Leu Thr GlnVal Gln Gln Arg Asn Leu Ile Ser Asn Leu Gln 210 215 220 Gln Ser Val AspAsp Thr Ser Leu Ala Ile Gln Arg Ile Lys Asn Asp 225 230 235 240 Phe GlnAsn Leu Gln Gln Val Phe Leu Gln Ala Lys Lys Asp Thr Asp 245 250 255 TrpLeu Lys Glu Lys Val Gln Ser Leu Gln Thr Leu Ala Ala Asn Asn 260 265 270Ser Ala Leu Ala Lys Ala Asn Asn Asp Thr Leu Glu Asp Met Asn Ser 275 280285 Gln Leu Ser Ser Phe Thr Gly Gln Met Asp Asn Ile Thr Thr Ile Ser 290295 300 Gln Ala Asn Glu Gln Ser Leu Lys Asp Leu Gln Asp Leu His Lys Asp305 310 315 320 Thr Glu Asn Arg Thr Ala Val Lys Phe Ser Gln Leu Glu GluArg Phe 325 330 335 Gln Val Phe Glu Thr Asp Ile Val Asn Ile Ile Ser AsnIle Ser Tyr 340 345 350 Thr Ala His His Leu Arg Thr Leu Thr Ser Asn LeuAsn Asp Val Arg 355 360 365 Thr Thr Cys Thr Asp Thr Leu Thr Arg His ThrAsp Asp Leu Thr Ser 370 375 380 Leu Asn Asn Thr Leu Val Asn Ile Arg LeuAsp Ser Ile Ser Leu Arg 385 390 395 400 Met Gln Gln Asp Met Met Arg SerLys Leu Asp Thr Glu Val Ala Asn 405 410 415 Leu Ser Val Val Met Glu GluMet Lys Leu Val Asp Ser Lys His Gly 420 425 430 Gln Leu Ile Lys Asn PheThr Ile Leu Gln Gly Pro Pro Gly Pro Arg 435 440 445 Gly Pro Lys Gly AspArg Gly Ser Gln Gly Pro Pro Gly Pro Thr Gly 450 455 460 Asn Lys Gly GlnLys Gly Glu Lys Gly Glu Pro Gly Pro Pro Gly Pro 465 470 475 480 Ala GlyGlu Arg Gly Thr Ile Gly Pro Val Gly Pro Pro Gly Glu Arg 485 490 495 GlySer Lys Gly Ser Lys Gly Ser Gln Gly Pro Lys Gly Ser Arg Gly 500 505 510Ser Pro Gly Lys Pro Gly Pro Gln Gly Pro Ser Gly Asp Pro Gly Pro 515 520525 Pro Gly Pro Pro Gly Lys Asp Gly Leu Pro Gly Pro Gln Gly Pro Pro 530535 540 Gly Phe Gln Gly Leu Gln Gly Thr Val Gly Glu Pro Gly Val Pro Gly545 550 555 560 Pro Arg Gly Leu Pro Gly Leu Pro Gly Val Pro Gly Met ProGly Pro 565 570 575 Lys Gly Pro Pro Gly Pro Pro Gly Pro Ser Gly Ala MetGlu Pro Leu 580 585 590 Ala Leu Gln Asn Glu Pro Thr Pro Ala Ser Glu ValAsn Gly Cys Pro 595 600 605 Pro His Trp Lys Asn Phe Thr Asp Lys Cys TyrTyr Phe Ser Leu Glu 610 615 620 Lys Glu Ile Phe Glu Asp Ala Lys Leu PheCys Glu Asp Lys Ser Ser 625 630 635 640 His Leu Val Phe Ile Asn Ser ArgGlu Glu Gln Gln Trp Ile Lys Lys 645 650 655 His Thr Val Gly Arg Glu SerHis Trp Ile Gly Leu Thr Asp Ser Glu 660 665 670 Gln Glu Ser Glu Trp LysTrp Leu Asp Gly Ser Pro Val Asp Tyr Lys 675 680 685 Asn Trp Lys Ala GlyGln Pro Asp Asn Trp Gly Ser Gly His Gly Pro 690 695 700 Gly Glu Asp CysAla Gly Leu Ile Tyr Ala Gly Gln Trp Asn Asp Phe 705 710 715 720 Gln CysAsp Glu Ile Asn Asn Phe Ile Cys Glu Lys Glu Arg Glu Ala 725 730 735 ValPro Ser Ser Ile Leu 740 5 27 PRT Artificial Sequence Consensus sequenceof three collectins which were reported heretofore. 5 Glu Asp Cys ValLeu Leu Leu Lys Asn Gly Gln Trp Asn Asp Val Pro 1 5 10 15 Cys Ser ThrSer His Leu Ala Val Cys Glu Phe 20 25 6 27 PRT Artificial SequenceModified Consensus Sequence of collectins Hybridizable with NovelCollectin. 6 Glu Lys Cys Val Glu Met Tyr Thr Asp Gly Lys Trp Asn Asp ArgAsn 1 5 10 15 Cys Leu Gln Ser Arg Leu Ala Ile Cys Glu Phe 20 25 7 24 DNAArtificial Sequence M13 Universal Primer Sequence for Sequencing. 7cgacgttgta aaacgacggc cagt 24 8 17 DNA Artificial Sequence M13 ReversePrimer Sequence for Sequencing. 8 caggaaaca gctatgac 17 9 21 DNAArtificial Sequence Sequence of a Reverse Primer for Screening a NovelCollectin. 9 caatctgatg agaaggtgat g 21 10 21 DNA Artificial SequenceSequence of a Forward Primer for Screening a Novel Collectin. 10acgaggggct ggatgggaca t 21 11 24 DNA Artificial Sequence Sequence of alambda gt11 Reverse Primer for Sequencing. 11 ttgacaccag accaactggt aatg24 12 24 DNA Artificial Sequence Sequence of a lambda gt11 ForwardPrimer for Sequencing. 12 ggtggcgacg actcctggag cccg 24 13 21 DNAArtificial Sequence Sequence of a Primer for Screening a NovelCollectin. 13 cgtgaaaatg aatggaagtg g 21 14 21 DNA Artificial SequenceSequence of a Primer for Screening a Novel Collectin. 14 ttttatccattgctgttcct c 21 15 21 DNA Artificial Sequence Sequence of a Primer forSequencing a Novel Collectin. 15 ctggcagtcc ccgaggtcca g 21 16 21 DNAArtificial Sequence Sequence of a Primer for Sequencing a NovelCollectin. 16 gctggtcccc ccggagagcg t 21 17 21 DNA Artificial SequenceSequence of a 1RC2 Primer for Cap Site Sequencing. 17 caaggtacgccacagcgtat g 21 18 20 DNA Artificial Sequence Sequence of a SyntheticTGP1 Primer for Cap Site Sequencing. 18 tcttcagttt ccctaatccc 20 19 21DNA Artificial Sequence Sequence of a 2RC2 Primer for Cap SiteSequencing. 19 gtacgccaca gcgtatgatg c 21 20 21 DNA Artificial SequenceSequence of a Synthetic TGP2 Primer for Cap Site Sequencing. 20cattcttgac aaacttcata g 21 21 21 DNA Artificial Sequence Sequence of aPrimer. 21 atcttgctgc agattcgtga c 21 22 15 DNA Artificial SequenceSequence of a lambda gt11 5′ Sequencing Primer. 22 gactcctgga gcccg 1523 2256 DNA Homo Sapiens CDS (74)..(1927) 23 ggggggacga cttcctcggctgcgcggcgc tcgcgcggag ctccccggcc ggcggtgcgt 60 ccccacggtc acc atg aaagac gac ttc gca gag gag gag gag gtg caa 109 Met Lys Asp Asp Phe Ala GluGlu Glu Glu Val Gln 1 5 10 tcc ttc ggt tac aag cgg ttt ggt att cag gaagga aca caa tgt acc 157 Ser Phe Gly Tyr Lys Arg Phe Gly Ile Gln Glu GlyThr Gln Cys Thr 15 20 25 aaa tgt aaa aat aac tgg gca ctg aag ttt tct atcata tta tta tac 205 Lys Cys Lys Asn Asn Trp Ala Leu Lys Phe Ser Ile IleLeu Leu Tyr 30 35 40 att ttg tgt gcc ttg cta aca atc aca gta gcc att ttggga tat aaa 253 Ile Leu Cys Ala Leu Leu Thr Ile Thr Val Ala Ile Leu GlyTyr Lys 45 50 55 60 gtt gta gag aaa atg gac aat gtc aca ggt ggc atg gaaaca tct cgc 301 Val Val Glu Lys Met Asp Asn Val Thr Gly Gly Met Glu ThrSer Arg 65 70 75 caa acc tat gat gac aag ctc aca gca gtg gaa agt gac ctgaaa aaa 349 Gln Thr Tyr Asp Asp Lys Leu Thr Ala Val Glu Ser Asp Leu LysLys 80 85 90 tta ggt gac caa act ggg aag aaa gct atc agc acc aac tca gaactc 397 Leu Gly Asp Gln Thr Gly Lys Lys Ala Ile Ser Thr Asn Ser Glu Leu95 100 105 tcc acc ttc aga tca gac att cta gat ctc cgt cag caa ctt cgtgag 445 Ser Thr Phe Arg Ser Asp Ile Leu Asp Leu Arg Gln Gln Leu Arg Glu110 115 120 att aca gaa aaa acc agc aag aac aag gat acg ctg gag aag ttacag 493 Ile Thr Glu Lys Thr Ser Lys Asn Lys Asp Thr Leu Glu Lys Leu Gln125 130 135 140 gcg agc ggg gat gct ctg gtg gac agg cag agt caa ttg aaagaa act 541 Ala Ser Gly Asp Ala Leu Val Asp Arg Gln Ser Gln Leu Lys GluThr 145 150 155 ttg gag aat aac tct ttc ctc atc acc act gta aac aaa accctc cag 589 Leu Glu Asn Asn Ser Phe Leu Ile Thr Thr Val Asn Lys Thr LeuGln 160 165 170 gcg tat aat ggc tat gtc acg aat ctg cag caa gat acc agcgtg ctc 637 Ala Tyr Asn Gly Tyr Val Thr Asn Leu Gln Gln Asp Thr Ser ValLeu 175 180 185 cag ggc aat ctg cag aac caa atg tat tct cat aat gtg gtcatc atg 685 Gln Gly Asn Leu Gln Asn Gln Met Tyr Ser His Asn Val Val IleMet 190 195 200 aac ctc aac aac ctg aac ctg acc cag gtg cag cag agg aacctc atc 733 Asn Leu Asn Asn Leu Asn Leu Thr Gln Val Gln Gln Arg Asn LeuIle 205 210 215 220 acg aat ctg cag cgg tct gtg gat gac aca agc cag gctatc cag cga 781 Thr Asn Leu Gln Arg Ser Val Asp Asp Thr Ser Gln Ala IleGln Arg 225 230 235 atc aag aac gac ttt caa aat ctg cag cag gtt ttt cttcaa gcc aag 829 Ile Lys Asn Asp Phe Gln Asn Leu Gln Gln Val Phe Leu GlnAla Lys 240 245 250 aag gac acg gat tgg ctg aag gag aaa gtg cag agc ttgcag acg ctg 877 Lys Asp Thr Asp Trp Leu Lys Glu Lys Val Gln Ser Leu GlnThr Leu 255 260 265 gct gcc aac aac tct gcg ttg gcc aaa gcc aac aac gacacc ctg gag 925 Ala Ala Asn Asn Ser Ala Leu Ala Lys Ala Asn Asn Asp ThrLeu Glu 270 275 280 gat atg aac agc cag ctc aac tca ttc aca ggt cag atggag aac atc 973 Asp Met Asn Ser Gln Leu Asn Ser Phe Thr Gly Gln Met GluAsn Ile 285 290 295 300 acc act atc tct caa gcc aac gag cag aac ctg aaagac ctg cag gac 1021 Thr Thr Ile Ser Gln Ala Asn Glu Gln Asn Leu Lys AspLeu Gln Asp 305 310 315 tta cac aaa gat gca gag aat aga aca gcc atc aagttc aac caa ctg 1069 Leu His Lys Asp Ala Glu Asn Arg Thr Ala Ile Lys PheAsn Gln Leu 320 325 330 gag gaa cgc ttc cag ctc ttt gag acg gat att gtgaac atc att agc 1117 Glu Glu Arg Phe Gln Leu Phe Glu Thr Asp Ile Val AsnIle Ile Ser 335 340 345 aat atc agt tac aca gcc cac cac ctg cgg acg ctgacc agc aat cta 1165 Asn Ile Ser Tyr Thr Ala His His Leu Arg Thr Leu ThrSer Asn Leu 350 355 360 aat gaa gtc agg acc act tgc aca gat acc ctt accaaa cac aca gat 1213 Asn Glu Val Arg Thr Thr Cys Thr Asp Thr Leu Thr LysHis Thr Asp 365 370 375 380 gat ctg acc tcc ttg aat aat acc ctg gcc aacatc cgt ttg gat tct 1261 Asp Leu Thr Ser Leu Asn Asn Thr Leu Ala Asn IleArg Leu Asp Ser 385 390 395 gtt tct ctc agg atg caa caa gat ttg atg aggtcg agg tta gac act 1309 Val Ser Leu Arg Met Gln Gln Asp Leu Met Arg SerArg Leu Asp Thr 400 405 410 gaa gta gcc aac tta tca gtg att atg gaa gaaatg aag cta gta gac 1357 Glu Val Ala Asn Leu Ser Val Ile Met Glu Glu MetLys Leu Val Asp 415 420 425 tcc aag cat ggt cag ctc atc aag aat ttt acaata cta caa ggt cca 1405 Ser Lys His Gly Gln Leu Ile Lys Asn Phe Thr IleLeu Gln Gly Pro 430 435 440 ccg ggc ccc agg ggt cca aga ggt gac aga ggatcc cag gga ccc cct 1453 Pro Gly Pro Arg Gly Pro Arg Gly Asp Arg Gly SerGln Gly Pro Pro 445 450 455 460 ggc cca act ggc aac aag gga cag aaa ggagag aag ggg gag cct gga 1501 Gly Pro Thr Gly Asn Lys Gly Gln Lys Gly GluLys Gly Glu Pro Gly 465 470 475 cca cct ggc cct gcg ggc tgc ccg cct cactgg aag aac ttc aca gac 1549 Pro Pro Gly Pro Ala Gly Cys Pro Pro His TrpLys Asn Phe Thr Asp 480 485 490 aaa tgc tac tat ttt tca gtt gag aaa gaaatt ttt gag gat gca aag 1597 Lys Cys Tyr Tyr Phe Ser Val Glu Lys Glu IlePhe Glu Asp Ala Lys 495 500 505 ctt ttc tgt gaa gac aag tct tca cat cttgtt ttc ata aac act aga 1645 Leu Phe Cys Glu Asp Lys Ser Ser His Leu ValPhe Ile Asn Thr Arg 510 515 520 gag gaa cag caa tgg ata aaa aaa cag atggta ggg aga gag agc cac 1693 Glu Glu Gln Gln Trp Ile Lys Lys Gln Met ValGly Arg Glu Ser His 525 530 535 540 tgg atc ggc ctc aca gac tca gag cgtgaa aat gaa tgg aag tgg ctg 1741 Trp Ile Gly Leu Thr Asp Ser Glu Arg GluAsn Glu Trp Lys Trp Leu 545 550 555 gat ggg aca tct cca gac tac aaa aattgg aaa gct gga cag ccg gat 1789 Asp Gly Thr Ser Pro Asp Tyr Lys Asn TrpLys Ala Gly Gln Pro Asp 560 565 570 aac tgg ggt cat ggc cat ggg cca ggagaa gac tgt gct ggg ttg att 1837 Asn Trp Gly His Gly His Gly Pro Gly GluAsp Cys Ala Gly Leu Ile 575 580 585 tat gct ggg cag tgg aac gat ttc caatgt gaa gac gtc aat aac ttc 1885 Tyr Ala Gly Gln Trp Asn Asp Phe Gln CysGlu Asp Val Asn Asn Phe 590 595 600 att tgc gaa aaa gac agg gag aca gtactg tca tct gca tta 1927 Ile Cys Glu Lys Asp Arg Glu Thr Val Leu Ser SerAla Leu 605 610 615 taacggactg tgatgggatc acatgagcaa attttcagctctcaaaggca aaggacactc 1987 ctttctaatt gcatcacctt ctcatcagat tgaaaaaaaaaaaagcactg aaaaccaatt 2047 actgaaaaaa aattgacagc tagtgttttt taccatccgtcattacccaa agacttggga 2107 actaaaatgt tccccagggt gatatgctga ttttcattgtgcacatggac tgaatcacat 2167 agattctcct ccgtcagtaa ccgtgcgatt atacaaattatgtcttccaa agtatggaac 2227 actccaatca gaaaaaggtt atcatcccg 2256 24 618PRT Homo Sapiens Deduced Amino Acid Sequence of Mutated Novel HumanScavenger Receptor from Nucleotide Sequence. 24 Met Lys Asp Asp Phe AlaGlu Glu Glu Glu Val Gln Ser Phe Gly Tyr 1 5 10 15 Lys Arg Phe Gly IleGln Glu Gly Thr Gln Cys Thr Lys Cys Lys Asn 20 25 30 Asn Trp Ala Leu LysPhe Ser Ile Ile Leu Leu Tyr Ile Leu Cys Ala 35 40 45 Leu Leu Thr Ile ThrVal Ala Ile Leu Gly Tyr Lys Val Val Glu Lys 50 55 60 Met Asp Asn Val ThrGly Gly Met Glu Thr Ser Arg Gln Thr Tyr Asp 65 70 75 80 Asp Lys Leu ThrAla Val Glu Ser Asp Leu Lys Lys Leu Gly Asp Gln 85 90 95 Thr Gly Lys LysAla Ile Ser Thr Asn Ser Glu Leu Ser Thr Phe Arg 100 105 110 Ser Asp IleLeu Asp Leu Arg Gln Gln Leu Arg Glu Ile Thr Glu Lys 115 120 125 Thr SerLys Asn Lys Asp Thr Leu Glu Lys Leu Gln Ala Ser Gly Asp 130 135 140 AlaLeu Val Asp Arg Gln Ser Gln Leu Lys Glu Thr Leu Glu Asn Asn 145 150 155160 Ser Phe Leu Ile Thr Thr Val Asn Lys Thr Leu Gln Ala Tyr Asn Gly 165170 175 Tyr Val Thr Asn Leu Gln Gln Asp Thr Ser Val Leu Gln Gly Asn Leu180 185 190 Gln Asn Gln Met Tyr Ser His Asn Val Val Ile Met Asn Leu AsnAsn 195 200 205 Leu Asn Leu Thr Gln Val Gln Gln Arg Asn Leu Ile Thr AsnLeu Gln 210 215 220 Arg Ser Val Asp Asp Thr Ser Gln Ala Ile Gln Arg IleLys Asn Asp 225 230 235 240 Phe Gln Asn Leu Gln Gln Val Phe Leu Gln AlaLys Lys Asp Thr Asp 245 250 255 Trp Leu Lys Glu Lys Val Gln Ser Leu GlnThr Leu Ala Ala Asn Asn 260 265 270 Ser Ala Leu Ala Lys Ala Asn Asn AspThr Leu Glu Asp Met Asn Ser 275 280 285 Gln Leu Asn Ser Phe Thr Gly GlnMet Glu Asn Ile Thr Thr Ile Ser 290 295 300 Gln Ala Asn Glu Gln Asn LeuLys Asp Leu Gln Asp Leu His Lys Asp 305 310 315 320 Ala Glu Asn Arg ThrAla Ile Lys Phe Asn Gln Leu Glu Glu Arg Phe 325 330 335 Gln Leu Phe GluThr Asp Ile Val Asn Ile Ile Ser Asn Ile Ser Tyr 340 345 350 Thr Ala HisHis Leu Arg Thr Leu Thr Ser Asn Leu Asn Glu Val Arg 355 360 365 Thr ThrCys Thr Asp Thr Leu Thr Lys His Thr Asp Asp Leu Thr Ser 370 375 380 LeuAsn Asn Thr Leu Ala Asn Ile Arg Leu Asp Ser Val Ser Leu Arg 385 390 395400 Met Gln Gln Asp Leu Met Arg Ser Arg Leu Asp Thr Glu Val Ala Asn 405410 415 Leu Ser Val Ile Met Glu Glu Met Lys Leu Val Asp Ser Lys His Gly420 425 430 Gln Leu Ile Lys Asn Phe Thr Ile Leu Gln Gly Pro Pro Gly ProArg 435 440 445 Gly Pro Arg Gly Asp Arg Gly Ser Gln Gly Pro Pro Gly ProThr Gly 450 455 460 Asn Lys Gly Gln Lys Gly Glu Lys Gly Glu Pro Gly ProPro Gly Pro 465 470 475 480 Ala Gly Cys Pro Pro His Trp Lys Asn Phe ThrAsp Lys Cys Tyr Tyr 485 490 495 Phe Ser Val Glu Lys Glu Ile Phe Glu AspAla Lys Leu Phe Cys Glu 500 505 510 Asp Lys Ser Ser His Leu Val Phe IleAsn Thr Arg Glu Glu Gln Gln 515 520 525 Trp Ile Lys Lys Gln Met Val GlyArg Glu Ser His Trp Ile Gly Leu 530 535 540 Thr Asp Ser Glu Arg Glu AsnGlu Trp Lys Trp Leu Asp Gly Thr Ser 545 550 555 560 Pro Asp Tyr Lys AsnTrp Lys Ala Gly Gln Pro Asp Asn Trp Gly His 565 570 575 Gly His Gly ProGly Glu Asp Cys Ala Gly Leu Ile Tyr Ala Gly Gln 580 585 590 Trp Asn AspPhe Gln Cys Glu Asp Val Asn Asn Phe Ile Cys Glu Lys 595 600 605 Asp ArgGlu Thr Val Leu Ser Ser Ala Leu 610 615 25 30 DNA Artificial SequenceSequence of a Primer for PCR Amplification of hSRCL-P1. 25 ccgctcgagcggtcaccatg aaagacgact 30 26 30 DNA Artificial Sequence Sequence of aPrimer for PCR Amplification of hSRCL-P1. 26 tccccgcggt aatgcagatgacagtactgt 30 27 35 DNA Artificial Sequence Sequence of a Primer for PCRAmplification of hSRCL-P1. 27 aatgcggccg caccatgaaa gacgacttcg cagag 3528 32 DNA Artificial Sequence Sequence of a Primer for PCR Amplificationof hSRCL-P1. 28 gctctagacc gcggtaatgc agatgacagt ac 32

What is claimed is:
 1. A protein comprising an amino acid sequenceconsisting of 742 amino acids set out in amino acid position 1 to 742 ofSEQ ID NO: 2, or a protein comprising an amino acid sequence set out inSEQ ID NO: 2 having deletion, substitution or addition of one or severalamino acids therein and having an equal property to that of the proteincomprising an amino acid sequence set out in amino acid position 1 to742 of SEQ ID NO: 2, or a derivative or a fragment thereof.
 2. Anisolated polynucleotide comprising a nucleotide sequence set out innucleotide position 74 to 2299 of SEQ ID NO: 1, a nucleotide sequenceencoding an amino acid sequence set out in amino acid position 1 to 742of SEQ ID NO: 2 or a fragment thereof, or a nucleotide sequence thathybridizes to any one of said nucleotide sequences or nucleotidesequences complementary thereto under a stringent condition and encodesa protein having an equal property to that of the protein comprising anamino acid sequence set out in amino acid position 1 to 742 of SEQ IDNO:
 2. 3. A protein comprising an amino acid sequence set out in aminoacid position 1 to 618 of SEQ ID NO: 24, or a protein comprising anamino acid sequence set out in SEQ ID NO: 24 having deletion,substitution or addition of one or several amino acids therein andhaving an equal property to that of the protein comprising an amino acidsequence set out in amino acid position 1 to 618 of SEQ ID NO: 24, or aderivative or a fragment thereof.
 4. An isolated polynucleotidecomprising a nucleotide sequence set out in nucleotide position 74 to1933 of SEQ ID NO: 23, a nucleotide sequence encoding an amino acidsequence set out in amino acid position 1 to 618 of SEQ ID NO: 24 or afragment thereof, or a nucleotide sequence that hybridizes to any one ofsaid nucleotide sequences or nucleotide sequences complementary theretounder a stringent condition and encodes a protein having an equalproperty to that of the protein comprising an amino acid sequence setout in amino acid position 1 to 618 of SEQ ID NO:
 24. 5. A proteincomprising an amino acid sequence consisting of 742 amino acids set outin amino acid position 1 to 742 of SEQ ID NO: 4, or a protein comprisingan amino acid sequence set out in SEQ ID NO: 2 having deletion,substitution or addition of one or several amino acids therein andhaving an equal property to that of the protein comprising an amino acidsequence set out in amino acid position 1 to 742 of SEQ ID NO: 2, or aderivative or a fragment thereof.
 6. An isolated polynucleotidecomprising a nucleotide sequence set out in nucleotide position 74 to2299 of SEQ ID NO: 3, a nucleotide sequence encoding an amino acidsequence set out in amino acid position 1 to 742 of SEQ ID NO: 2 or afragment thereof, or a nucleotide sequence that hybridizes to any one ofsaid nucleotide sequences or nucleotide sequences complementary theretounder a stringent condition and encodes a protein having an equalproperty to that of the protein comprising an amino acid sequence setout in amino acid position 1 to 742 of SEQ ID NO:
 2. 7. A vectorcomprising a polynucleotide according to claim 2, 4 or
 6. 8. Atransformed cell carrying a polynucleotide according to claim 2, 4 or 6in a manner to allow the expression.
 9. A method for the production of aprotein which comprises the steps of culturing a cell transformed withthe polynucleotide according to claim 2 or 4, and collecting thusproduced hSRCL-P1 protein.
 10. A method for the production of a proteinwhich comprises the steps of culturing a cell transformed with thepolynucleotide according to claim 6, and collecting thus producedmSRCL-P1 protein.
 11. The method according to claim 9 or 10 wherein saidcell is Escherichia coli, an animal cell or an insect cell.
 12. Atransgenic non-human animal having an altered expression level ofSRCL-P1 gene.
 13. The transgenic non-human animal according to claim 12wherein said SRCL-P1 gene is cDNA, genomic DNA or synthesized DNAencoding SRCL-P1.
 14. The transgenic non-human animal according to claim13 wherein the expression level is altered by causing the mutation at agene expression regulatory site.
 15. A knockout mouse wherein a functionof mSRCL-P1 gene is deficient.
 16. An antibody to the protein accordingto claim 1, 3 or 5, or a fragment thereof.
 17. The antibody according toclaim 16, which is a polyclonal antibody, a monoclonal antibody or apeptide antibody.
 18. A method for the production of a monoclonalantibody to the protein or the fragment thereof according to claim 1, 3or 5 which comprises administering the protein or a fragment thereofaccording to claim 1, 3 or 5 to a warm-blooded animal other than human,selecting the animal that exhibits an antibody titer, collecting aspleen or a lymph node from the animal, fusing antibody-producing cellscontained therein with myeloma cells to prepare a hybridoma thatproduces a monoclonal antibody.
 19. A method for quantitativelydetermining an SRCL-P1 protein or a fragment thereof on the basis of animmunological binding between the antibody according to claim 16 or 17and the SRCL-P1 protein or a fragment thereof.
 20. A method fordetecting an SRCL-P1 protein or a fragment thereof on the basis of animmunological binding between the antibody according to claim 16 or 17and the SRCL-P1 protein or a fragment thereof.
 21. An agonist thatstimulates an activity of the protein according to claim 1, 3 or
 5. 22.An antagonist that inhibits an activity or the activation of the proteinaccording to claim 1, 3 or
 5. 23. A method for screening a drug whereinthe protein according to claim 1, 3 or 5 is used.
 24. A drug which isobtained by the method for the screening according to claim
 23. 25. Amethod for screening a drug for the treatment of a pathological stateinvolved in the accumulation of oxidized LDL, which comprises the stepof identifying a candidate drug for the treatment of a pathologicalstate involved in the accumulation of oxidized LDL by an inhibitoryability of the candidate drug toward the binding between the proteinaccording to claim 1, 3 or 5 and oxidized LDL, which is evaluated bycomparing the amount of binding between the protein and oxidized LDL inthe presence and absence of the candidate drug.
 26. A drug obtained bythe method for the screening according to claim
 25. 27. A method for thetreatment of a pathological state involved in the accumulation ofoxidized LDL, which comprises the step of inhibiting the binding betweenan SRCL-P1 protein or a fragment thereof and oxidized LDL using the drugaccording to claim
 26. 28. A pharmaceutical composition for thetreatment of a pathological state involved in the accumulation ofoxidized LDL comprising the drug according to claim
 26. 29. A method forscreening a drug for the treatment of a pathological state involved inthe binding of AGE to cells, which comprises the step of identifying acandidate drug for the treatment of a pathological state involved in thebinding of AGE to cells by an inhibitory ability of the candidate drugtoward the binding between the protein according to claim 1, 3 or 5 andAGE, which is evaluated by comparing the amount of binding between theprotein and AGE in the presence and absence of the candidate drug.
 30. Adrug obtained by the method for the screening according to claim
 29. 31.A method for the treatment of a pathological state involved in thebinding of AGE to cells, which comprises the step of inhibiting thebinding between an SRCL-P1 protein or a fragment thereof and AGE usingthe drug according to claim
 30. 32. A pharmaceutical composition for thetreatment of a pathological state involved in the binding of AGE tocells comprising the drug according to claim 30.